Adverse outcomes in chronic myeloid leukemia patients treated with tyrosine kinase inhibitors: Follow-up of patients diagnosed 2002-2017 in a complete coverage and nationwide agnostic register study.

Tyrosine kinase inhibitors (TKIs) have profoundly improved the clinical outcome for patients with chronic myeloid leukemia (CML), but their overall survival is still subnormal and the treatment is associated with adverse events. In a large cohort-study, we assessed the morbidity in 1328 Swedish CML chronic phase patients diagnosed 2002-2017 and treated with TKIs, as compared to that in carefully matched control individuals. Several Swedish patient registers with near-complete nationwide coverage were utilized for data acquisition. Median follow-up was 6 (IQR, 3-10) years with a total follow-up of 8510 person-years for the full cohort. Among 670 analyzed disease categories, the patient cohort showed a significantly increased risk in 142 while, strikingly, no category was more common in controls. Increased incidence rate ratios/IRR (95% CI) for more severe events among patients included acute myocardial infarction (AMI) 2.0 (1.5-2.6), heart failure 2.6 (2.2-3.2), pneumonia 2.8 (2.3-3.5), and unspecified sepsis 3.5 (2.6-4.7). When comparing patients on 2nd generation TKIs vs. imatinib in a within-cohort analysis, nilotinib generated elevated IRRs for AMI (2.9; 1.5-5.6) and chronic ischemic heart disease (2.2; 1.2-3.9), dasatinib for pleural effusion (11.6; 7.6-17.7) and infectious complications, for example, acute upper respiratory infections (3.0; 1.4-6.0). Our extensive real-world data reveal significant risk increases of severe morbidity in TKI-treated CML patients, as compared to matched controls, particularly for 2nd generation TKIs. Whether this increased morbidity may also translate into increased mortality, thus preventing CML patients to achieve a normalized overall survival, needs to be further explored.

Successful tyrosine kinase inhibitor discontinuation outside clinical trials - data from the population-based Swedish chronic myeloid leukaemia registry.

Clinical trials show that tyrosine kinase inhibitor (TKI) treatment can be discontinued in selected patients with chronic myeloid leukaemia (CML). Although updated CML guidelines support such procedure in clinical routine, data on TKI stopping outside clinical trials are limited. In this retrospective study utilising the Swedish CML registry, we examined TKI discontinuation in a population-based setting. Out of 584 patients diagnosed with chronic-phase CML (CML-CP) in 2007-2012, 548 had evaluable information on TKI discontinuation. With a median follow-up of nine years from diagnosis, 128 (23%) discontinued TKI therapy (≥1 month) due to achieving a DMR (deep molecular response) and 107 (20%) due to other causes (adverse events, allogeneic stem cell transplant, pregnancy, etc). Among those stopping in DMR, 49% re-initiated TKI treatment (median time to restart 4·8 months). In all, 38 patients stopped TKI within a clinical study and 90 outside a study. After 24 months 41·1% of patients discontinuing outside a study had re-initiated TKI treatment. TKI treatment duration pre-stop was longer and proportion treated with second-generation TKI slightly higher outside studies, conceivably affecting the clinical outcome. In summary we show that TKI discontinuation in CML in clinical practice is common and feasible and may be just as successful as when performed within a clinical trial.

A large observational study of patients with primary immune thrombocytopenia receiving romiplostim in European clinical practice.

OBJECTIVE: Romiplostim has maintained long-term platelet counts in patients with immune thrombocytopenia (ITP) for up to 5 yr in clinical studies. This prospective observational study aimed to describe romiplostim utilisation and outcomes in European clinical practice. METHODS: Adults with primary ITP who received romiplostim in routine care were eligible. RESULTS: Three-hundred and forty patients were eligible for analysis, of whom 299 (88%) completed the 2-yr observation period. The median age was 62 yr, with 43% of patients aged ≥65 yr, and two-thirds of patients initiated romiplostim before splenectomy. The median average weekly dose of romiplostim was 2.8 µg/kg. The median baseline platelet count was 20 × 109 /L, which increased after 2 wk of romiplostim treatment and remained >50 × 109 /L thereafter. After romiplostim initiation, there was a decrease in rates of grade ≥3 bleeding events (from 12 to 2 per 100 patient-years) and ITP-related hospitalisations (from 87 to 33 per 100 patient-years). The rate of thrombotic events was 2 per 100 patient-years, and bone marrow fibrosis occurred in two patients. CONCLUSIONS: Romiplostim dosing, effectiveness and safety in an unselected real-world ITP population seemed comparable with that observed in clinical studies.

Advanced phase chronic myeloid leukaemia (CML) in the tyrosine kinase inhibitor era - a report from the Swedish CML register.

OBJECTIVES: The primary goal in management of chronic phase (CP) chronic myeloid leukaemia (CML) is to prevent disease progression to accelerated phase (AP) or blast crisis (BC). We have evaluated progression rates in a decentralised healthcare setting and characterised patients progressing to AP/BC on TKI treatment. METHODS: Using data from the Swedish CML register, we identified CP-CML patients diagnosed 2007-2011 who progressed to AP/BC within 2 yrs from diagnosis (n = 18) as well as patients diagnosed in advanced phase during 2007-2012 (n = 36) from a total of 544 newly diagnosed CML cases. We evaluated baseline characteristics, progression rates, outcome and adherence to guidelines for monitoring and treatment. RESULTS: The cumulative progression rate at 2 yrs was 4.3%. All 18 progression cases had been treated with imatinib, and six progressed within 6 months. High-risk EUTOS score was associated to a higher risk of progression. Insufficient cytogenetic and/or molecular monitoring was found in 33%. Median survival after transformation during TKI treatment was 1.4 yrs. In those presenting with BC and AP, median survival was 1.6 yrs and not reached, respectively. CONCLUSION: In this population-based setting, progression rates appear comparable to that reported from clinical trials, with similar dismal patient outcome. Improved adherence to CML guidelines may minimise the risk of disease progression.

IL1RAP antibodies block IL-1-induced expansion of candidate CML stem cells and mediate cell killing in xenograft models.

Chronic myeloid leukemia (CML) is currently treated with tyrosine kinase inhibitors, but these do not effectively eliminate the CML stem cells. As a consequence, CML stem cells persist and cause relapse in most patients upon drug discontinuation. Furthermore, no effective therapy exists for the advanced stages of the disease. Interleukin-1 receptor accessory protein (IL1RAP; IL1R3) is a coreceptor of interleukin-1 receptor type 1 and has been found upregulated on CML stem cells. Here, we show that primitive (CD34+CD38-) CML cells, in contrast to corresponding normal cells, express a functional interleukin-1 (IL-1) receptor complex and respond with NF-κB activation and marked proliferation in response to IL-1. IL1RAP antibodies that inhibit IL-1 signaling could block these effects. In vivo administration of IL1RAP antibodies in mice transplanted with chronic and blast phase CML cells resulted in therapeutic effects mediated by murine effector cells. These results provide novel insights into the role of IL1RAP in CML and a strong rationale for the development of an IL1RAP antibody therapy to target residual CML stem cells.

Altered cytokine levels in pediatric ITP.

Immune thrombocytopenia (ITP) is an autoimmune disease where platelets are destroyed prematurely. In the majority of children, the disease resolves, but in some, it becomes chronic. Cytokines are important mediators of the immune response and are known to be dysregulated in autoimmune diseases. Therefore, our aim was to investigate differences in plasma levels of cytokines between children with ITP and healthy controls. We had two cohorts of children: one Swedish with 18 children with ITP and seven healthy children and a second Chinese one with 58 children with ITP and 30 healthy children. Plasma levels of chemokine (C-X3-C motif) ligand 1 (CX3CL1), transforming growth factor ß1 (TGF-ß1), and interleukin 22 (IL-22) were analyzed in both cohorts using enzyme-linked immunosorbent assays (ELISAs). We found lower plasma levels of TGF-ß1 and elevated levels of CX3CL1 and IL-22 in children with ITP compared with controls in both the Swedish and the Chinese cohort. In conclusion, all three cytokines differ between pediatric ITP and healthy controls and may, therefore, be potential biomarkers for the disease.

Second malignancies following treatment of chronic myeloid leukaemia in the tyrosine kinase inhibitor era.

Given that tyrosine kinase inhibitors (TKIs) have dramatically improved the survival of patients with chronic myeloid leukaemia (CML), we were interested in examining the possible risk of long-term adverse events, such as the emergence of other neoplasms. Therefore, we studied the development of second malignancies in 868 patients diagnosed with CML between 2002 and 2011 using the Swedish CML register, cross-linked to the Swedish Cancer register. With a median follow-up of 3·7 (range 0-9·9) years, 65 (7·5%) patients developed 75 second malignancies (non-haematological), 52 of which were of the invasive type. Compared to expected rates in the background population, the risk of second malignancies was higher in the CML cohort, with a standardized incidence ratio (SIR) of 1·52 (95% CI 1·13-1·99). The SIR before and after the second year following diagnosis of CML was 1·58 and 1·47, respectively. Among specific cancer types, gastrointestinal and nose and throat cancer were significantly increased. Founded on a population-based material, our results indicate that CML patients treated in the TKI era are at an increased risk of developing a second malignancy, with indications that this risk may more likely be linked to CML itself rather than to the TKI treatment.

Tyrosine kinase inhibitor usage, treatment outcome, and prognostic scores in CML: report from the population-based Swedish CML registry.

Clinical management guidelines on malignant disorders are generally based on data from clinical trials with selected patient cohorts. In Sweden, more than 95% of all patients diagnosed with chronic myeloid leukemia (CML) are reported to the national CML registry, providing unique possibilities to compile population-based information. This report is based on registry data from 2002 to 2010, when a total of 779 patients (425 men, 354 women; median age, 60 years) were diagnosed with CML (93% chronic, 5% accelerated, and 2% blastic phase) corresponding to an annual incidence of 0.9/100,000. In 2002, approximately half of the patients received a tyrosine kinase inhibitor as initial therapy, a proportion that increased to 94% for younger (<70 years) and 79% for older (>80 years) patients during 2007-2009. With a median follow-up of 61 months, the relative survival at 5 years was close to 1.0 for patients younger than 60 years and 0.9 for those aged 60 to 80 years, but only 0.6 for those older than 80 years. At 12 months, 3% had progressed to accelerated or blastic phase. Sokal, but not European Treatment and Outcome Study, high-risk scores were significantly linked to inferior overall and relative survival. Patients living in university vs nonuniversity catchment areas more often received tyrosine kinase inhibitors up front but showed comparable survival.

MicroRNA regulate immune pathways in T-cells in multiple sclerosis (MS).

BACKGROUND: MicroRNA are small noncoding RNA molecules that are involved in the control of gene expression. To investigate the role of microRNA in multiple sclerosis (MS), we performed genome-wide expression analyses of mRNA and microRNA in T-cells from MS patients and controls. METHODS: Heparin-anticoagulated peripheral blood was collected from MS-patients and healthy controls followed by isolation of T-cells. MicroRNA and RNA from T-cells was prepared and hybridized to Affymetrix miR 2.0 array and Affymetrix U133Plus 2.0 Human Genome array (Santa Clara, CA), respectively. Verifications were performed with real-time polymerase chain reaction (PCR) and enzyme-linked immunosorbent assay (ELISA). RESULTS: We identified 2,452 differentially expressed genes and 21 differentially expressed microRNA between MS patients and controls. By Kolmogorov-Smirnov test, 20 of 21 differentially expressed microRNA were shown to affect the expression of their target genes, many of which were involved in the immune system. Tumor necrosis factor ligand superfamily member 14 (TNFSF14) was a microRNA target gene significantly decreased in MS. The differential expression of mir-494, mir-197 and the predicted microRNA target gene TNFSF14 was verified by real-time PCR and ELISA. CONCLUSION: These findings indicate that microRNA may be important regulatory molecules in T-cells in MS.

Differences in gene expression and cytokine levels between newly diagnosed and chronic pediatric ITP.

Immune thrombocytopenia (ITP) is an autoimmune disease where platelets are destroyed prematurely. In the majority of children the disease resolves, but in some it becomes chronic. To investigate whether these 2 phases of the disease are molecularly similar or separate entities we performed DNA microarray analysis (GEO accession number: GSE46922) of T-cells from newly diagnosed children and children with chronic ITP. We found complete separation of the gene expression profiles between the 2 phases of the disease. Furthermore, the gene expression levels of several cytokines differed between the 2 phases of the disease. This was also reflected in plasma with increased levels of interleukin (IL)-16 and TNF-related weak inducer of apoptosis and lower levels of IL-4 in newly diagnosed compared with chronic ITP. Thus, our data indicate that chronic ITP in childhood is a separate disease entity, dissimilar in many aspects to the newly diagnosed phase.

MicroRNA regulate immunological pathways in T-cells in immune thrombocytopenia (ITP).

MicroRNA are small noncoding RNA molecules that regulate gene expression. To investigate the role of microRNA in immune thrombocytopenia (ITP), we performed genome-wide expression analyses of mRNA and microRNA in T cells from ITP patients and controls. We identified 1915 regulated genes and 22 regulated microRNA that differed between ITP patients and controls. Seventeen of the 22 regulated microRNA were linked to changes in target gene expression; 57 of these target genes were associated with the immune system, eg, T-cell activation and regulation of immunoglobulin production. CXCL13 and IL-21 were two microRNA target genes significantly increased in ITP. We could demonstrate increased plasma levels of CXCL13 and others have reported increased plasma levels of interleukin-21 in ITP. Thus, regulated microRNA were significantly associated with both gene and protein expression of molecules in immunological pathways. We suggest that microRNA may be important regulatory molecules involved in the loss of tolerance in ITP.

Serum levels of LIGHT in MS.

BACKGROUND: Recently, a polymorphism in the LIGHT gene was shown to increase the risk of multiple sclerosis (MS) in a genome-wide association study (GWAS). OBJECTIVE: Our aim was to investigate if serum levels of LIGHT were affected by this polymorphism and by the disease itself. METHODS: Serum levels of LIGHT were investigated in four cohorts; 1) MS (n = 159) and controls (n = 160) in relation to rs1077667 genotype; 2) MS at relapse (n = 30) vs. healthy controls (n = 26); 3) MS (n = 27) vs. other neurological disease (OND, n = 33); and 4) MS patients before and after one year of treatment with natalizumab (n = 30). RESULTS: Carriers of the GG genotype had the lowest serum levels of LIGHT (p=0.02). Serum levels of LIGHT were increased in MS at relapse in two separate cohorts: vs. healthy controls (p=0.00005) and vs. remission (p=0.00006), other neurological disease (OND) (p=0.002) and OND with signs of inflammation (iOND; p=0.00005). Furthermore, serum levels of LIGHT were decreased by natalizumab treatment (p=0.001). CONCLUSION: Soluble LIGHT is an inhibitor of T-cell activation and GG carriers of rs1077667, with the highest risk for MS, had the lowest serum levels. The increased levels of LIGHT at times of increased MS activity suggest that soluble LIGHT is protective and may act to limit inflammation.

MS risk genes are transcriptionally regulated in CSF leukocytes at relapse.

BACKGROUND: Infiltrating T-helper cells, cytotoxic T-cells, B-cells and monocytes are thought to mediate the damage to myelin, oligodendrocytes and axons in multiple sclerosis (MS), which results in progressive disability. OBJECTIVE: The objective of this paper is to explore gene expression profiles of leukocytes in the cerebrospinal fluid (CSF) compartment of MS patients during relapse. METHODS: Global gene expression was analyzed by DNA microarray analysis of cells in CSF from MS patients and controls, and verifications were performed with real-time polymerase chain reaction (PCR) and enzyme-linked immunosorbent assay (ELISA). RESULTS: Fifty percent of the recently described risk genes for MS and 28% of non-risk genes were differently expressed in MS patients compared to controls (χ(2)-test, p=7.7 × 10(-5)). Genes involved in T- and NK-cell processes were up-regulated, and genes involved in processes targeting innate immunity or B-cells were down-regulated in MS. Increased expression of EDN1 and CXCL11 and decreased expression of HMOX1 was verified with real-time PCR and increased expression of CXCL13 was verified with ELISA in CSF. CONCLUSION: DNA microarray analysis is useful in identifying differently expressed genes in CSF leukocytes, which may be important in MS in vivo. Our findings suggest that many of the risk genes for MS are differently expressed in the disease-mediating leukocytes that penetrate the blood-brain barrier.

Differential expression of T-cell genes in blood and bone marrow between ITP patients and controls.

Primary immune thrombocytopenia (ITP) is an autoimmune disease characterised by premature platelet destruction in spleen, liver and bone marrow and a diminished production of platelets. T-cells are important in all forms of autoimmunity including ITP; however, very little is known about T-cells in organs where platelets are destroyed. Our aim was to investigate differences in gene expression in peripheral blood-derived T-cells and bone marrow-derived T-cells between ITP patients and controls. T-cells and subsequent RNA were isolated from blood and bone marrow from chronic ITP patients and healthy controls followed by DNA microarray analysis. There were 1554 differentially expressed genes in peripheral blood-derived T-cells and 976 in bone marrow-derived T-cells between ITP patients and controls and three genes were verified with real-time PCR. Using Gene Ontology functional enrichment analysis we found that genes involved in growth, development, migration, chemotaxis, adhesion and apoptosis were enriched in bone marrow-derived T-cells in ITP. Immune-related genes involved in T-helper cell differentiation, T-cell chemotaxis, migration, immunoglobulin-mediated immune response and classical and alternative pathway complement activation were also enriched in bone marrow-derived T-cells in ITP. Only 213 T-cell genes were differentially expressed in both blood and bone marrow between ITP patients and controls. In conclusion, our findings show that genes involved in major pathophysiologic pathways in ITP such as T-helper cell differentiation, autoantibody response and complement activation are altered in bone marrow-derived T-cells in ITP patients compared with controls. This further supports the concept that bone marrow is an important compartment in ITP.

Secondary hyperparathyroidism but stable bone-mineral density in patients with chronic myeloid leukemia treated with imatinib.

Imatinib is currently the standard treatment for chronic myeloid leukemia(CML). Previous studies have shown that imatinib affects bone metabolism in CML patients. However, these effects are not well-studied prospectively. The authors studied bone-mineral density (BMD) and bone metabolism in 17 CML patients and matched controls in 2007 and now repeated the analyses prospectively in 2011. All CML patients were in complete cytogenetic remission during this 4-year period and treated with 400 mg imatinib q.d. (n 5 15) or 600 mg imatinib q.d. (n 5 2). Mean treatment duration was 102 months (range 69­129) in 2011. The authors found that serum levels of parathyroid hormone increased significantly in the patients between 2007 and 2011, and seven out of 17 patients had secondary hyperparathyroidism in 2011. However, the mean areal and volumetric BMDs were stable in the CML patients over the 4-year-observation period. Moreover, the CML patients had significantly higher volumetric BMD in the cortical compartment when compared with controls in 2011 and 2007. Thus, despite a high incidence of secondary hyperparathyroidism,there were no signs of osteoporosis or osteomalacia in imatinib-treated CML patients as suggested earlier.

Imatinib inhibits proliferation of human mesenchymal stem cells and promotes early but not late osteoblast differentiation in vitro.

Altered bone metabolism has been reported in patients with chronic myeloid leukemia treated with the tyrosine kinase inhibitor imatinib. Several studies have shown that imatinib inhibits the differentiation and activity of osteoclasts in vitro, whereas the effects of imatinib on osteoblast differentiation are less clear. In this study osteoblast differentiation was induced in human mesenchymal stem cells (hMSCs) by treatment with bone morphogenetic protein 2 in vitro. Imatinib inhibited proliferation of hMSCs in a dose-dependent manner. Even though imatinib promoted early osteoblast differentiation assessed by alkaline phosphate activity, mineralization measured by Alizarin Red staining (ARS) was reduced by imatinib. Moreover, the inhibitory effect of imatinib on mineralization was most prominent at low concentrations of imatinib. When we measured the relative mRNA expression levels of Runx2, we found that Runx2 expression was higher in imatinib-treated (5 µM) cultures at early time points during differentiation. On the other hand, the expression of Osterix late during differentiation was lower in imatinib-treated (5 µM) cultures, corresponding to the ARS results. Thus, the effect of imatinib on osteoblast differentiation is not only dependent on the drug concentration, but indeed also on the maturation stage of the cells. This finding might partly explain why previous studies on the effects of imatinib osteoblast differentiation have shown different results.

Increased number of B-cells in the red pulp of the spleen in ITP.

Platelets are targeted by autoantibodies and destroyed in the reticuloendothelial system in the spleen, liver and bone marrow in patients with immune thrombocytopenia (ITP). Other mechanisms such as destruction by cytotoxic T-cells and defective production of platelets in the bone marrow also exist. Splenectomy normalizes the platelet count in 70% of ITP patients, however, precious little is known about the spleen in this disease. Our aim was therefore to investigate the splenic morphology and especially the number and localization of splenic leukocytes in patients with ITP and controls and to evaluate factors predicting outcome of splenectomy. Spleen sections from 29 ITP patients and 11 individuals splenectomized due to trauma were analyzed by immunohistochemistry. All except one of the ITP patients had a normalized platelet count 12 months after splenectomy and the platelet count was inversely correlated with age. ITP patients had an increased number of B-cells in the red pulp. The number of white pulp B-cells and number of T-cells in both compartments was unchanged. In conclusion, B-cells are increased in the red pulp of the spleen and together with cytotoxic T-cells, helper T-cells and macrophages line the sinusoids enabling the immunological attack on platelets in ITP.