Course Of Chronic Myeloid Leukemia Research Articles

Somatic Mutations in Commonly Mutated Genes in Myeloid Malignancies May Preexist or Arise in the Course of Chronic Myeloid Leukemia - Different Scenarios of Progression Revealed By Targeted Next-Generation Sequencing

Chronic myeloid leukemia (CML) is currently a true chronic disease for majority of patients who achieve durable remission with tyrosine kinase inhibitors (TKI) and remain in chronic phase (CML-CP) for several years. However, a number of patients develop TKI resistance and may progress to blastic phase (CML-BP) which represent major therapeutic challenge. Although CML-BP resembles acute myeloid leukemia (AML) in many aspects, including selected genetic aberrations, pathogenesis of CML progression to acute phase is not fully understood. Therefore there is a strong rationale for studying underlying mechanisms of progression to CML-BP.To gain comprehensive insight into genetic background of CML progression, we performed targeted high-throughput sequencing of sequential DNA samples from patients experiencing a treatment failure and progression to CML-BP. We inquired whether gene mutations previously described in human malignancies (with special focus on genes involved in leukemogenesis) were gained during progression or if they existed already at diagnosis. Sequential samples were collected from 5 patients, who progressed to CML-BP, despite TKI therapy and for whom samples were available from both diagnosis and progression. Roche NimbleGen SeqCap EZ custom-capture was used to acquire exonic sequences of approximately 1000 cancer-related genes, comprising genes from commercially available cancer panels (such as Illumina TrueSight Cancer, NimbleGen Comprehensive Cancer Panel) and also genes altered in hematological malignancies as reported in current literature. Common variants (>1%) gathered in ESP6500 and 1000 genomes projects and our internal exome database were filtered out.Patient CML-1 was diagnosed in CML-BP and relapsed within 13 months despite treatment with 2 TKIs. Both at the diagnosis and progression, a DNMT3A p. P799T mutation was detected with similar allele frequency. Patient CML-2 was diagnosed in CML-CP, underwent alloHSCT within 6 months from diagnosis and achieved short-term remission. However, within few months she relapsed with lymphoid BP and despite short-lived responses to combined chemotherapy and TKI, she developed 2nd BP and 3rd BP with BCR-ABL1 p. Y253H and p. T315I mutations, respectively. Strikingly, p. R320* RUNX1 mutation was present at all four time points (diagnosis in CML-CP and all three BPs), though at diagnosis frequency of this mutation was approx. 1%. Patient CML-3 wasdiagnosed in CML-BP and was treated with TKIs plus chemotherapy. After 25 months patient developed resistance and progressed. We detected IDH1 p. R132S mutation, typical for AML, exclusively in the BP sample from progression, while it was absent in diagnostic sample. Patient CML-4 was diagnosed in CML-CP, developed resistance to 3 TKIs and finally progressed to CML-BP after 5 years. P. Y183C IDH1 mutation was detected in the BP sample. Patient CML-5 was diagnosed in CP and despite treatment with TKI, progressed to CML-BP within 3,5 years. P. R139L and p. R166L RUNX1 mutations were detected only in the sample from CML-BP. All detected mutations were confirmed independently by Sanger sequencing or deep amplicon sequencing for low frequency variants. Detected mutations are summarized in table 1.Our analysis of sequential samples from CML patients proves that mutations in genes commonly mutated in myeloid malignancies (DNMT3A, IDH1, RUNX1) may be preexisting or may arise during progression, independently of BCR-ABL1 mutation. With regard to preexisting mutations, this may lead to clonal evolution of the disease. Importantly, such preexisting mutations could have been missed in the previous studies, presented or published, employing next-generation sequencing strategy, since most of those studies used algorithms to detect newly gained aberrations in CML-BP as compared to CML-CP.Table 1Mutations detected in patients experiencing TKI resistance and progression with no detectable BCR-ABL1 mutations.PatientTime to progression [months]MutationsCML-113DNMT3A p. P799TpreexistingCML-29RUNX1 p. R320*preexistingCML-325IDH1 p. R132SacquiredCML-463IDH1 p. Y183CacquiredCML-541RUNX1 p. R139L RUNX1 p. R166Lacquired acquired DisclosuresNo relevant conflicts of interest to declare.

First report of imatinib measurement in hair: Method development and preliminary evaluation of the relation between hair and plasma concentrations with therapeutic response in chronic myeloid leukemia

BackgroundImatinib (IM) is a first choice drug for treatment of chronic myeloid leukemia (CML), with a widely accepted concentration threshold of 1000ng/ml being used as a target for therapeutic drug monitoring. Once adherence to the pharmacotherapeutic regimen is of paramount importance during the long treatment course of CML, the measurement of hair IM concentrations could be a surrogate of the patient's exposure to the drug. MethodsIM was extracted from a 5mg hair sample by a liquid–liquid extraction with ethyl acetate, and IM-d8 was used as internal standard (IS). After evaporation, and reconstitution in acetonitrile, the extract was injected into a LC–MS/MS system. Compounds were eluted on a C8 column in isocratic mode. IM and IS were identified in positive electrospray ionization mode using ion transitions of m/z 494.5>394.5 and 503.0>394.3 respectively. The method was applied to 102 paired hair and samples obtained from CML patients. Treatment response was evaluated according to the European LeukemiaNet recommendations. ResultsThe assay was validated in the concentration range of 0.5–25ng/mg, with intra- and inter-assay imprecisions of <13.1% and <9.3%, respectively. The limits of quantification and detection were 0.5 and 0.15ng/mg, respectively. Median hair IM concentrations are significantly smaller in patients with therapeutic failure when compared with patients with partial or optimal response (4.63 vs. 7.93, p=0.040), the same trend presented by median plasma IM concentrations (629.5 vs. 1084.8, p=0.009). An IM hair concentration below 5.8ng/mg has 83% sensibility and 70% specificity to identify patients with therapeutic failure. ConclusionsA fast, sensitive, and selective LC–MS/MS method allowing quantification of IM in hair samples was developed and validated. CML patients with therapeutic failure had significantly lower hair IM concentrations when compared with patients with optimal response. These preliminary findings may support the use of hair as a matrix for IM monitoring in clinical settings, with significant logistic advantages over the collection of venous blood, particularly in developing countries.

Major Route Additional Chromosomal Aberrations (ACA) Precede Increase of Blasts in CML: An Analysis of the German CML-Studies III and IIIA

▪During the course of chronic myeloid leukemia (CML) progression to blast crisis (BC) is thought to be caused by genetic instability such as cytogenetic aberrations in addition to the translocation t(9;22)(q34;q11). We have shown previously that major route ACA indicate an unfavorable outcome (Fabarius et al., Blood 2011). We now investigate whether there is a correlation in time between appearance of major route ACA and increase in blast count.Methods: Cytogenetic data and blast count in the peripheral blood were available from 1,290 CML patients recruited to the German CML-studies III (621 patients) and IIIa (669 patients) from January 1995 to January 2004. Treatments were interferon-alpha-based or related allogeneic stem cell transplantation (HSCT). Presence of ACA and major route ACA was considered as a time-dependent covariate. Multivariate proportional hazards models were estimated taking Euro CML score, study III vs. IIIa and stem cell transplantability into account. Cumulative incidences of blast increases were calculated starting at the date of the first ACA or major route ACA, respectively, regarding death as a competing risk. Patients were censored at the date of HSCT with an unrelated donor.Results: 1,287 patients were evaluable with median observation times of 13 and 12 years and a 10-year survival of 48% and 61% in CML studies III and IIIa, respectively. 258 patients progressed to BC with a cumulative 10-year incidence of 20%. 195 patients displayed ACA during the course of disease. 45 patients (15.7%) showed ACA already at diagnosis. 44 patients showed unbalanced minor route, 29 balanced minor route aberrations, 23 -Y. 109 patients showed major route aberrations including 10 with other prior ACA.In a multivariate analysis on 1,257 patients, patients with ACA had a hazard ratio (HR) for a blast increase of between 2.0-2.2 (p<0.001) for blast increases to ≥1%, ≥5%, ≥10%, ≥15%, ≥ 20% and ≥30% compared with patients without ACA (Table). When the same model was performed for major route ACA only at any time during disease, HRs of 2.2-2.7 (p<0.001) were found. For ACA without major route ACA HRs were 1.6-2.1 (p<0.001). In the multivariate analyses of major route ACA vs. no major route ACA a blast increase of 1-5% after diagnosis of major route ACA seems already indicative of progression.5 years after the diagnosis of any ACA the cumulative incidence for a blast increase was 30% (95%- confidence interval (CI): 23-38%), of a major route ACA 40% (95%- CI: 28-49%). The 6-year probability of death without blast increase was 10%. 14 additional patients received an unrelated transplant of which 6 died.We conclude that ACA, particularly major route ACA, precede an increase of blasts. Major route ACA have to be considered as a prognostic indicator for disease progression at any time.Table 1Blast increase toHR (univariate): ACA vs. no ACAHR(multivariate)*: ACA vs. no ACAHR (univariate): major route ACA vs. no major route ACAHR (multivariate)*: major route ACA vs. no major route ACA≥30%2.4092.1392.6462.203≥20%2.4132.1442.6562.211≥15%2.4152.1612.8682.426≥10%2.4162.1602.7992.357≥5%2.2862.0472.7192.278≥1%2.2091.9993.1712.684*adjusted to Euro-Score, study (III vs. IIIa) and transplantability DisclosuresSaussele:ARIAD: Honoraria; BMS: Honoraria, Other: Travel grant, Research Funding; Pfizer: Honoraria, Other: Travel grant; Novartis Pharma: Honoraria, Other: Travel grant, Research Funding. Haferlach:MLL Munich Leukemia Laboratory: Employment, Equity Ownership. Scheid:Janssen-Cilag: Honoraria, Membership on an entity's Board of Directors or advisory committees; Celgene: Honoraria, Membership on an entity's Board of Directors or advisory committees. Baerlocher:Geron Corporation: Research Funding; Novartis: Research Funding. Schnittger:MLL Munich Leukemia Laboratory: Employment, Equity Ownership. Müller:BMS: Consultancy, Honoraria, Research Funding; Ariad: Consultancy, Honoraria, Research Funding; Novartis: Consultancy, Honoraria, Research Funding. Hochhaus:ARIAD: Honoraria, Research Funding; Pfizer: Honoraria, Research Funding; Bristol-Myers Squibb: Honoraria, Research Funding; Novartis: Honoraria, Research Funding. Pfirrmann:BMS: Consultancy, Honoraria; Novartis Pharma: Consultancy, Honoraria. Baccarani:Bristol-Myers Squibb: Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; NOVARTIS: Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; ARIAD Pharmaceuticals, Inc.: Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; PFIZER: Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau. Hehlmann:BMS: Consultancy; Novartis Pharma: Research Funding.

BCL2 gene polymorphisms and splicing variants in chronic myeloid leukemia

Recent data suggest that constitutional genetic variation in the antiapoptotic BCL2 gene could be associated with the susceptibility to develop chronic myeloid leukemia (CML) and the clinical outcome in several hematological malignancies. The present study examines whether BCL2 single nucleotide polymorphisms (SNPs) predispose to CML or may potentially influence the disease characteristics at diagnosis. Notably, no association was observed between the four candidate BCL2 SNPs and the risk of developing CML. Instead, the 4777C>A (rs2279115) and the 5735A>G (rs1801018) SNPs were significantly associated with the disease risk profile as determined by the Sokal score. We found that such polymorphisms correlated with the expression of BCL2 alternative splicing transcripts (BCL2-α, BCL2-β) in healthy donors, but not in CML patients, although the relative levels of BCL2 mRNA splicing variants were shown to change during the clinical course of CML. Our findings suggest that BCL2 polymorphisms could influence the clinical features of CML patients at diagnosis. However, the pathogenic mechanisms involved in such association remain to be ascertained.

Safety and efficacy of imatinib in CML over a period of 10 years: data from the randomized CML-study IV.

Tyrosine kinase inhibitors (TKI) have changed the natural course of chronic myeloid leukemia (CML). With the advent of second-generation TKI safety and efficacy issues have gained interest. The randomized CML - Study IV was used for a long-term evaluation of imatinib (IM). 1503 patients have received IM, 1379 IM monotherapy. After a median observation of 7.1 years, 965 patients (64%) still received IM. At 10 years, progression-free survival was 82%, overall survival 84%, 59% achieved MR(5), 72% MR(4.5), 81% MR(4), 89% major molecular remission and 92% MR(2) (molecular equivalent to complete cytogenetic remission). All response levels were reached faster with IM800 mg except MR(5). Eight-year probabilities of adverse drug reactions (ADR) were 76%, of grades 3-4 22%, of non-hematologic 73%, and of hematologic 28%. More ADR were observed with IM800 mg and IM400 mg plus interferon α (IFN). Most patients had their first ADR early with decreasing frequency later on. No new late toxicity was observed. ADR to IM are frequent, but mostly mild and manageable, also with IM 800 mg and IM 400 mg+IFN. The deep molecular response rates indicate that most patients are candidates for IM discontinuation. After 10 years, IM continues to be an excellent initial choice for most patients with CML.

Bone marrow fibrosis may be an effective independent predictor of the ‘TKI drug response level’ in chronic myeloid leukemia

ObjectivesThe aim of this study was to assess bone marrow (BM) fibrosis and dysplasia in chronic myeloid leukemia (CML) patients receiving the first-generation tyrosine kinase inhibitor (TKI), imatinib, or second-generation TKIs, dasatinib, and nilotinib. We further investigated whether CML under TKI is associated with dysplastic BM changes during the clinicopathological course of the disease.MethodsIn total, pre-treatment BM paraffin blocks of biopsy specimens were available for 41 adult patients diagnosed with chronic phase CML. Post-treatment BM aspirate clot and core biopsy samples were reviewed for fibrosis and dyshematopoiesis.ResultsOverall, 13 (31.7%) patients achieved a complete cytogenetic response with imatinib treatment, with no events. In 25 patients, imatinib was discontinued owing to primary or secondary resistance. In patients with initial dysmyelopoiesis, the rate of BM fibrosis was 82.4 versus 47.6% for other patient groups (P = 0.02). Overall, 24 patients with newly diagnosed CML showed marrow fibrosis, among which 19 (79.1%) had imatinib resistance. However, only 5 out of 15 patients (33.5%) without marrow fibrosis had imatinib resistance (P = 0.08).DiscussionOur findings indicate that BM fibrosis is an independent predictor of the ‘TKI drug response level’ in CML and support its inclusion as a critical pathobiological parameter for decision-making with regard to TKI drug selection de novo, calculation of prognosis at the onset of disease, and monitoring response to TKI in the long-term disease course of CML.

Decisions Taken in Children and Adolescents with Chronic Myeloid Leukemia (CML) at Failure of Imatinib Treatment

Abstract Background: CML is rare in pediatrics; &lt;20 patients (pts) younger than 18 yrs are diagnosed in Germany annually. So far only imatinib (IMA) is fully licensed for treatment of pediatric CML while off-label use of 2nd generation tyrosine kinase inhibitors (TKIs) dasatinib (DASA) or nilotinib (NILO) is possible and is reimbursed by most German insurance companies by decision on an individual basis. Clinical features of CML at diagnosis differ age-dependently in minors in comparison to older adults [Pemmaraju N et al, Haematologica 2012, 97:1029; Kalmanti L et al, Ann Hematol 2014, 93:71] and also prominent differences could be demonstrated in controlled pediatric trials (CML-PAED I/II; French trial IV) when analyzing stage of disease (more advanced in pediatrics), spleen size (larger in pediatrics), and leukocyte number (higher in pediatrics) [Suttorp M et al, Hematology ASH Educ Program 2010:368; Millot F et al, J Clin Oncol 2011, 29:2817]. Also in children the up-front treatment of CML with IMA resulted in postponing stem cell transplantation (SCT). We here report on the course of CML in a greater cohort of pediatric and adolescent pts treated up-front by IMA as well as decisions taken at TKI failure due to intolerance or resistance. Methods: At diagnosis pts were enrolled prospectively into the ongoing trial CML-PAED-II and treated with a standardized IMA dose (300/400/500 mg/sqm in chronic phase (CP); in accelerated phase (AP); in blast crisis (BC); respectively) under regular monitoring at three months intervals (Suttorp M et al, Bone Marrow Transplant 2008). Response rates (hematological, cytogenetic, molecular) and side effects were monitored following ELN guidelines. At treatment failure pts were switched to either a 2nd generation TKI or underwent HLA-matched (10/10 alleles) related or unrelated SCT on an individual basis [de la Fuente J et al, Br J Haematol 2014 Jun 30 epub ahead of print]. Results: As of April 2014 129 pts (57 fem, 72 male; mean age at diagnosis: 11.2 yrs; range: 1–17 yrs) could be analyzed. Stage of disease was CML-CP (n=120), CML-AP (n=5), and CML-BC (n=4; 2 myeloid / 2 lymphoid). With a median follow-up of 40 months (range: 1-96 mo) 77 pts (60%) of 129 pts still are on IMA while 48 pts experienced failure. In this situation only 18/48 opted for transplantation, while 30 pts were switched to a 2nd generation TKI (see Figure). 27/30 pts received DASA and 3/30 pts NILO as 2nd line TKI treatment. In this cohort, 16 pts also failed 2nd line TKI treatment (13 pts while on DASA and 3 pts while on NILO). After DASA failure, 8 pts went on to allogeneic SCT and 5 were treated with NILO as 3rd line treatment whereas after NILO failure 1 pt received DASA and 2 pts were transplanted. Out of 28 pts undergoing SCT 5 pts have died. Death was caused by transplant related complications (bleeding, infection, GvHD) in 3 pts (2 pts after failing 1st line TKI / 1 pt after 2nd line failure) and relapse of CML in 2 pts (diagnosed in CML-BC and transplanted after failing 1st line TKI). Out of 81 pts on ongoing IMA as 1st line treatment, 4 pts achieved PCR negativity for a minimum period of 2 years and have stopped the TKI maintaining complete molecular remission for so far 3, 5, 20, 46 mo, respectively [Moser O et al, Ped Blood Cancer 2014, May 9 epub ahead of print]. Conclusion: Side effects of IMA are well tolerated by the majority of pediatric pts. At failure of IMA only one third of the pts opted for SCT while the majority of treating physicians selected 2nd generation TKIs. For 2nd line treatment DASA was preferred to NILO. However, the precise role of these 2nd generation TKIs which presently are tested in activated / ongoing pediatric phase I/II trials still has to be clarified in minors. Figure 1 Figure 1. Disclosures No relevant conflicts of interest to declare.

A multi-state model approach for prediction in chronic myeloid leukaemia.

Multi-state models support prediction in medicine. With different states of disease, chronic myeloid leukaemia (CML) is particularly suited for the application of multi-state models. In this article, we tried to find a model for CML that allows predicting the prevalence of three different states (initial state of disease, remission and progression) in dependence on treatment, adjusted for age, sex and risk score. Based on the German CML Study IV, one of the largest randomised studies in CML, the model was able to represent the known effects of age and risk score on the probabilities of remission and progression. Patients achieving a major molecular remission had a better chance of surviving without progression, but this effect was not significant. Comparing treatments, patient of the high-dose arm had the greatest chance to be in the state "remission" at 5 years but did not seem to have an advantage considering "progression". The proposed illness-death model can be useful for predicting the course of CML based on the patient's individual covariates (trial registration: this is an explorative analysis of ClinicalTrials.gov Identifier: NCT00055874).

Molecular monitoring and mutations in chronic myeloid leukemia: how to get the most out of your tyrosine kinase inhibitor.

The course of chronic myeloid leukemia (CML) and the response to treatment with tyrosine kinase inhibitors (TKIs) are best monitored and assessed using two molecular tests: the first is real-time quantitative reverse transcription-polymerase chain reaction (RQ-PCR), which measures the size of residual disease that is expressed as BCR-ABL1% (the ratio between BCR-ABL1 and a control gene) and the other is mutational analysis by Sanger sequencing, which checks for the presence of BCR-ABL1 kinase domain point mutations. Both tests are technically demanding and require a high level of specialization and standardization. RQ-PCR, when performed on a regular basis, allows for the defining of molecular response (MR) levels as log reduction from a standardized baseline: major molecular response (MMR or MR(3)) that is the best predictor of survival; and the deeper molecular response (MR(4), MR(4.5), and MR(5)) that is necessary to enroll a patient in a trial aiming at treatment-free remission (TFR). Mutational analysis, to be performed in case of failure or warning by Sanger sequencing, allows for screening of the BCR-ABL1 kinase domain for mutations conferring resistance to TKIs. Since different mutations have different degrees of sensitivity to each of the currently available TKI, the knowledge of BCR-ABL1 kinase domain-mutation status is necessary for subsequent treatment choice. Optimal patient management requires that MR and mutational information be rationally interpreted at both the technical and at the biologic level, and put into context-therapeutic decisions also take into account other factors, such as age, comorbidities, side effects, compliance, and treatment-related complications.

Kinase-Independent Mechanisms of Resistance of Leukemia Stem Cells to Tyrosine Kinase Inhibitors

Tyrosine kinase inhibitors such as imatinib mesylate have changed the clinical course of chronic myeloid leukemia; however, the observation that these inhibitors do not target the leukemia stem cell implies that patients need to maintain lifelong therapy. The mechanism of this phenomenon is unclear: the question of whether tyrosine kinase inhibitors are inactive inside leukemia stem cells or whether leukemia stem cells do not require breakpoint cluster region (Bcr)-Abl signaling is currently under debate. Herein, I propose an alternative model: perhaps the leukemia stem cell requires Bcr-Abl, but is dependent on its kinase-independent functions. Kinases such as epidermal growth factor receptor and Janus kinase 2 possess kinase-independent roles in regulation of gene expression; it is worth investigating whether Bcr-Abl has similar functions. Mechanistically, Bcr-Abl is able to activate the Ras, phosphatidylinositol 3-kinase/Akt, and/or the Src-kinase Hck/Stat5 pathways in a scaffolding-dependent manner. Whereas the scaffolding activity of Bcr-Abl with Grb2 is dependent on autophosphorylation, kinases such as Hck can use Bcr-Abl as substrate, inducing phosphorylation of Y177 to enable scaffolding ability in the absence of Bcr-Abl catalytic activity. It is worth investigating whether leukemia stem cells exclusively express kinases that are able to use Bcr-Abl as substrate. A kinase-independent role for Bcr-Abl in leukemia stem cells would imply that drugs that target Bcr-Abl's scaffolding ability or its DNA-binding ability should be used in conjunction with current therapeutic regimens to increase their efficacy and eradicate the stem cells of chronic myeloid leukemia.

Expression of transketolase-like gene 1 (TKTL1) depends on disease phase in patients with chronic myeloid leukaemia (CML)

Overexpression of transketolase-like gene 1 (TKTL1) on RNA and protein level has been linked to tumour progression, metastasis and unfavourable patient outcome in many solid tumours. Chronic myeloid leukaemia (CML) cells show metabolic characteristics resembling deviations observed in TKTL1 overexpressing solid tumour cells. We therefore sought to evaluate TKTL1 gene expression in different phases of CML. A total of 120 peripheral blood samples from 69 patients in various phases of CML and 21 healthy individuals were investigated. TKTL1 expression levels were determined by real-time quantitative polymerase chain reaction using LightCycler technology and normalised against beta-glucuronidase expression. A significantly lower TKTL1 expression was found in chronic phase (CP) CML patients compared to healthy controls. Lowest expression levels were observed in patients during blast crisis (BC). Baseline TKTL1 expression in CP patients did not have value in prognostication of subsequent favourable or dismal outcome. Further, more mature granulocytes showed significantly higher TKTL1 expression compared to immature CD34+ and CD34-/CD33+ cells both in healthy controls and in CML patients. TKTL1 expression levels appear to decline in the course of CML with lowest levels during BC. A potential reason is a shift of TKTL1-high-expressing mature granulocytes towards TKTL1-low-expressing immature cells and blasts.

Differential Expression of BIRC Family Genes In The Course Of Chronic Myeloid Leukemia – BIRC3 and BIRC8 As Potential New Candidates To Identify Disease Progression

Although majority of chronic myeloid leukemia (CML) patients benefit from targeted therapy, there is an unmet need to identify as early as possible patients who develop resistance to tyrosine kinase inhibitors (TKI) and/or patients who progress to blastic phase (CML-BP). Searching for potential candidates of disease progression we have focused on BIRC (baculoviral IAP repeat-containing) genes expression in various stages of CML. This family includes eight functionally- and structurally-related proteins, most of them are believed to serve as endogenous inhibitors of apoptosis. Overexpression of various BIRC genes has been associated with cancer progression, multidrug resistance, poor prognosis and short survival in several types of neoplasms including hematological malignancies. In CML so far expression of BIRC5 (encoding survivin) and BIRC4 (encoding XIAP) has been associated with progression of the disease. However, there is no data on the role of other BIRC members in myeloproliferative diseases.To study the expression of BIRC genes in CML we employed RT-qPCR following MIQE guidelines. We analyzed sequential samples of peripheral blood leukocytes obtained from CML patients at various stages of the disease. Initially we looked at the samples from patients in chronic phase (CML-CP): at the diagnosis and after development of TKI resistance (confirmed as a loss of cytogenetic response, n=5). Then we analyzed samples from patients who progressed either to accelerated phase (CML-AccPh) or to blastic phase (CML-BP) (n=6). Among eight BIRC genes we observed significant decrease in BIRC3 (encoding cIAP2) and BIRC8 (encoding ILP2) expression. This was associated both with TKI resistance and with progression of CML to accelerated or blastic phase. Simultaneously, we observed marked increase in BIRC5 expression in samples from CML-AccPh and BP as compared to chronic phase (as was previously shown by others) but we observed no significant difference in BIRC5 expression between CML-CP diagnostic and TKI-resistant samples. Expression of BIRC1 (NAIP), BIRC2 (cIAP1), BIRC4 (XIAP), BIRC6 (BRUCE) and BIRC7 (LIVIN) was comparable in sequential samples from CML-CP and CML-BC and was not related to TKI-resistance.We verified these results by comparing larger group of patients in either CML-CP at the diagnosis prior to any treatment (n=15) or CML-BP (n=11). To compare the expression of BIRC family in CML to normal hematopoietic cells, we included also cDNA from healthy blood donors (n=10). In accordance with paired samples analysis, we observed downregulation of BIRC3 and BIRC8 expression in CML-BP (as compared to CML-CP and healthy blood donors), while BIRC5 was upregulated in CML-BP patients (as compared to CML-CP and healthy blood donors). There was no difference in expression of other BIRC family members.In conclusion, this is the first comprehensive analysis of the expression of all eight BIRC genes in the course of CML. In addition to the previously described upregulation of BIRC5, we observed significant downregulation of BIRC3 and BIRC8 associated with TKI-resistance and also with progression to accelerated or blastic phase. Recently Rossi D. et al. (Blood 2012;119: 2854-62) described potential role of disruption of BIRC3 (through mutation or gene deletion) in resistance to fludarabine in chronic lymphocytic leukemia. Together with our results this shows that current view linking BIRC genes upregulation with tumor progression and drug resistance may not be true for all of BIRC genes. Our results suggest novel and unexpected role of BIRC3 and BIRC8 in the clonal evolution of CML and open a new area for further exploration of the role of BIRC in CML progression. Disclosures:No relevant conflicts of interest to declare.

Current management of chronic myeloid leukemia with tyrosine kinase inhibitors.

The clinical outcomes and survival of tyrosine kinase inhibitor (TKI)-treated patients with chronic myeloid leukemia (CML) have been significantly improved. The aim of this editorial is to outline critical steps of TKI administration practices during the long-term clinical course of CML based on data obtained from randomized clinical trials and international recommendations. The efficacy of TKI treatment, TKI side effects, off-target complications, and long-term morbidities due to both the disease and the drug are common arguments in the management of CML. Complete hematological response, early complete cytogenetic response, faster major molecular response, and deeper, more durable molecular responses (MR4, MR4.5, MR5) are the ultimate goals for TKI-receiving patients with CML.Conflict of interest:None declared.

Homozygosity for killer immunoglobin-like receptor haplotype A predicts complete molecular response to treatment with tyrosine kinase inhibitors in chronic myeloid leukemia patients

Several recent reports suggest a possible role for killer immunoglobulin-like receptors (KIR) in the onset of chronic myeloid leukemia (CML) and response to therapy with tyrosine kinase inhibitors (TKIs). To explore this hypothesis, we studied KIRs and their human leukocyte antigen class I ligands in 59 consecutive patients with chronic-phase CML (mean age, 53 years; range, 23-81 years) and a group of 121 healthy control participants belonging to the same ethnic group as the patients. The 2-year cumulative incidence of complete molecular response, obtained after a median of 27 months (range, 4-52 months), was 51.2%. An increased frequency of the activating receptor KIR2DS1 (pm = 0.05) and a reduced frequency of the KIR-ligand combination KIR2DS2/2DL2 absent/C1 present (pm = 0.001) were significantly associated with CML. Moreover, KIR repertoires in patients appeared to influence response to TKI therapy. Homozygosity for KIR haplotype A (pm = 0.01), a decreased frequency of the inhibitory KIR gene KIR2DL2 (pm = 0.02), and low numbers of inhibitory KIR genes (pm = 0.05) were all significantly associated with achievement of complete molecular remission. These data suggest that a decrease in properly stimulated and activated NK cells might contribute to the occurrence of CML and indicate homozygosity for KIR haplotype A as a promising immunogenetic marker of complete molecular response that could help clinicians decide whether to withdraw treatment in patients with CML.

The Musashi 2 mRNA Expression in the Course of CML.

Abstract 2552The Musashi (MSI) gene family members, MSI1 and MSI2, represent an evolutionarily conserved family of RNA-binding proteins that regulate mRNA translation through binding in their N-termini. High levels of MSI2 protein are associated with increased cell proliferation, decreased cell maturation, more aggressive hematologic malignancy diseases and worse clinical prognosis. Recently obtained data pointed to MSI2 playing an important role in acute myeloid leukemia (AML) and in deadly blast crisis of chronic myeloid leukemia (CML) (Ito et al. 2010 Nature 5; 466).In this study we screened the level of MSI2 mRNA in 49 patients in different phases of CML and with different response to therapy – 18 patients at diagnosis (DG), 5 in major molecular response (MMR), 4 in complete molecular response (CMR), 2 after bone marrow transplantation (BMT), 10 in hematology relaps (HR), 6 in accelerrated phase (AP), and 4 in blast crisis (BC), and in 6 healthy donors. The level of MSI2 mRNA was quantified by real-time reverse-transcriptase-polymerase chain reaction using in-house designed specific primers and TaqMan probe and normalized to B2M endogenous control. Expression ratios were calculated by ΔΔCt method, and the differences between groups were statistically evaluated using Mann Whitney test.We detected MSI2 expression in all samples. The median expression of mRNA MSI2 in patients at DG was 1,43 (0,33–3,28), in MMR 0,52 (0,20–0,62), in CMR 0,37 (0,30–0,63), after BMT 1,28 (1,02–1,54), in HR 0,41 (0,16–0,58), in AP 3,78 (1,94–13,69), in BC 15,17 (2,61–28,15). MSI2 expression was statistically up-regulated in patients in advanced phases of CML (AP, BC) when compared with patients in CP (P<0.0001). The difference between patients in DG and remaining patients in CP was also statistically significant (P= 0,0006). No correlation of MSI2 expression level in DG patients with their responsiveness to treatment, BCR-ABL transcript level or survival was found. No significant differences were observed among groups of patients in MMR, CMR, HR, and after BMT.In addition, in order to check whether MSI2 expression level can serve as a marker of CML progression we also retrospectively screened kinetics of MSI2 transcript in 5 CML patients monitored on average 27 months (18–48). During this period, 3 patients developed HR, 1 patient AP and 1 BC. In BC patient the MSI2 transcript level increased with progression of CML in accordance with the increase of leucocytes and BCR-ABL transcript level. In 1 patient with a rising AP BCR-ABL levels remained constant compared to sevenfold increase of the MSI2 transcript level. On the other hand in HR patients we detected a constant or even decreasing level of MSI2 transcript regardless of the increase of leucocytes and BCR-ABL.In summary, our results confirm the association of high MSI2 mRNA level with advanced phases of CML and indicate that increase of MSI2 mRNA level may serve as a valuable marker of advanced phases of CML. In particular for CML patients with constantly high level of BCR-ABL mRNA the monitoring of MSI2 level can be important tool for early recognition of CML progression. Potential contributions of MSI2 to leukemic pathogenesis and its regulation in CML progression remain unknown.Grant support: NT/12392-4 IGA MZ-CR. Disclosures:No relevant conflicts of interest to declare.

Survival Analysis of Additional Chromosomal Aberrations in Philadelphia Chromosome Positive and Negative Cells: A Single Institutional

Abstract 2523 Background:Chronic myeloid leukemia (CML) is a clonal stem cell malignancy associated with the Philadelphia chromosome, t(9;22)(q34q31)/BCR-ABL gene fusion. Additional cytogenetic abnormalities have been known to emerge in Philadelphia (Ph) positive cells (additional Ph+ clones) or Ph negative cells (Ph- clones) at diagnosis, or during or post tyrosine kinase inhibitor (TKI) therapy. Many studies have elucidated that the presence of some Ph+ clones were frequently associated with disease progression, while presence of Ph- clones were unrelated to disease outcome. A majority of studies have shown a waxing and waning of Ph- clonality in response to therapy. However, there remains no solid data regarding overall survival comparing Ph+ and Ph- clones over a long term period. This study focuses on the observation of clonal evolution in various phases of CML, and the relationship to overall survival, disease resistance, kinase domain mutation (KDM) and progression to accelerated or blast phase in patients with CML.Materials and Methods: Data from 318 patients who were diagnosed with CML was retrieved from Moffitt Cancer Center during January 1990-December 2010. Patients are divided into three groups based on the presence of additional ph+ clone (A), ph- clone (B) and absence of both (C). Clinicopathologic results including initial diagnosis date, nature and frequency of clone, copy of clone at karyotyping, disease status in response to treatment and overall outcome were documented and statistically analyzed. All patients with Ph- and Ph+ clones have been treated with TKIs since 2001. Median overall survival and median disease progression survival were compared between conal ph- and clonal ph+ group by the log-rank test. Survival curves were generated using the Kaplan–Meier method. All reported P values are two-sided. Cox Regression (multivariate analysis) was also performed for time to progression. Results:Of 318 (average age 57, range: 19 to 89, M:F=1:1), 17 carried ph- clones (5.3%) and 41 showed additional ph+ clones (12.9%) and the rest (258) lacked additional ph+ or ph- clones. Additional clonal cytogenetic aberrations were random with the most frequent occurrence of trisomy 8 (7 of 17 ph- and 14 of 41 ph+ clones, respectively) and isochromosome 17q (4 in ph+ clone only). There is a higher rate of transformation to accelerated/blast phase in Ph+ additional clones (36.6%,15 of 41) as compared to Ph- additional clone (17.6%, 3 of 17) and Ph+ without additional clones(11.1%, 29 of 260) (p=0.015). The overall median survival is shorter in patients with ph+ clones (133.4 months) than in those with ph- clones (172 months) (p<0.005). KDM were observed in 27 of 110 tested patients (24.5%), and many of them fell into the Ph+ clone group (10 of 22 tested) and fewer into ph- clone group (3 of 10 tested) (p<0.005). Statistical analysis proved that there is a higher rate of transformation to accelerated/blast phase in the presence of a KDM (p<0.005). Of note, most of those with ph- clones had only one episode (59%) with 41% having more than one occurrence and lasting from 6–58 months (average 21.1 months), while in those 41 patients with Ph+ clone; a subset (18 of 41, 43.9%) showed a long-lasting ph+ clone over months (ranging from 2 months to 70 months, average 14.5 months) and the remaining only occurred one occasion (23 of 41, 56.1%). Multivariate analysis for disease progression demonstrated statistical significance with regards to both the ph+ clone and kinase mutation categories (p=0.003 and 0.034). Conclusion:Most Ph+ or Ph- clones are occasionally observed during the long course of CML, with a minor subset of clones showing persistency, especially in Ph- ones. The presence of an additional Ph+ clone is correlated with disease progression, drug resistance and shorter overall survival in comparison to a Ph- clone. A relatively long overall survival is observed in patients with ph- clones. Ph- clones were often identified during disease remission regardless of frequency of clonal copy or clonal persistence. The outcome of patients with concurrent Ph+ and Ph- clones was dictated by the Ph+ clone. Disclosures:No relevant conflicts of interest to declare.

The Gene Expressions of hOCT1 and ABCB1 Change During the Course of CML in Relation to Imatinib Therapy

Abstract 2495 Background.The intracellular concentration of imatinib (IM) in patients with chronic myeloid leukemia (CML) is supposed to be influenced by the expression of its main cellular transporters, hOCT1 (influx) and ABCB1 (efflux). The assessment of the genes expression may be potentially important in clinical practice to effectively manage the therapy. Our recent results showed the necessity to cautiously interpret results from gene expression measurements of both transporters as their detected mRNA levels are affected by the proportion of different cell types in the sample analyzed (Racil et al. 2010 Am J Hem 85:525, Racil et al. 2011 Leuk & Lymph 52:331). Aims. In this report, we aimed to comprehensively assess differences in the expression of hOCT1 and ABCB1 in total leukocytes of peripheral blood (PB) in relation to the blood cell lineage in CML patients with different responses to IM. Methods. Kruskall Wallis’s and Dunn’s multiple comparison tests were applied to calculate differences in transcript levels of hOCT1 and ABCB1 in patients at diagnosis (Dg=43), in major molecular response (MMR=27), complete molecular response (BCR-ABL log 4.5 reduction; CMR4.5 =15), therapy failure (TF=13), accelerated phase (AP=12) and in 75 healthy controls. CMR4.5 is defined here as either a detectable disease ≤0.0032% BCR-ABLIS or as undetectable by nested PCR. TF is defined as non CCgR achievement. Additionally, we used the Spearman’s correlation test to investigate relationship between expressions and percentage of immature cells and neutrophils in patients with non-physiological blood count (Dg and AP). In patients with normal blood count (CMR4.5, MMR, TF), we calculated correlation with BCR-ABL transcript level. Finally, we performed in vitro experiments with BCR-ABL negative (SKM-1, MOLM-13) and positive cell lines (K562, MOLM-7) treated with IM to study its effect on ABCB1 expression. Results. We found a significantly lower expression of hOCT1 and ABCB1 at Dg (P<0.001) and in AP (P<0.001 and P<0.01, respectively) in comparison to controls. The lower expression probably depends indirectly on the immature cells count (hOCT1 r = −0.4850, P=0.0002; ABCB1 r = −0.6451, P< 0.0001). Interestingly, while a significant positive correlation of neutrophil count was found with hOCT1 mRNA levels (r=0.6090, P< 0.0001), no correlation was observed with ABCB1. In patients with physiological blood count, we observed significantly elevated expression of ABCB1 in MMR and CMR4.5 in comparison to controls (P< 0.01). As MMR and CMR4.5 samples are characterized by the BCR-ABL transcript level <0.1%, this result led us to perform in vitro experiments to test the effect of IM on ABCB1 expression in BCR-ABL negative cell lines showing an elevated expression after incubation with IM. We assume this effect to be a natural defense of BCR-ABL negative cells to get rid of IM. By contrast, we found a drop in ABCB1 expression after BCR-ABL positive cell lines incubation with IM, suggesting inhibitory effects of IM. A significantly reduced expression of hOCT1 was found in TF in comparison to MMR and CMR4.5 (P< 0.01). These samples did not differ in blood count, but we observed a significant negative correlation of hOCT1 with BCR-ABL transcript levels (r= − 0.5117; P< 0.0001). Conclusion. Cell composition in PB changes during the CML treatment with IM which influences the measurement of hOCT1 and ABCB1 mRNA levels in total leukocytes. However, hOCT1 expression was found to be significantly lower in patients who failed to achieve CCgR but had normal blood count. Interestingly, our data suggest that ABCB1 is differently expressed in BCR-ABL positive and negative cells due to the inhibitory and enhancing effects of IM, respectively. Supported by IGA NT11555 and MZOUHKT2005 Disclosures:Machova Polakova:Novartis: travel grant. Ondrackova:BMS: travel grant.

Bilateral Hip Pain Caused by Adductor Pyomyositis as the Initial Presentation of Chronic Myeloid Leukemia in a 17-Year-Old Child

Pyomyositis is a pyogenic muscular tissue infection mainly occurring in immunocompromised patients. Chronic myeloid leukemia (CML) accounts for only 2-3% of cases of childhood leukemia. Herein, we report on a 17-year-old male with bilateral hip pain caused by adductor pyomyositis before beginning the treatment course of CML. CML was diagnosed by bone marrow chromosome study and was treated initially with imatinib but switched to hydroxyurea 5 days later because of poor cytoreduction response. Subsequently, white blood cell counts decreased gradually; however, the hyperleukocytosis condition resolved very slowly again until we switched back to imatinib use on the 40(th) day of hospitalization. Pyomyositis was diagnosed by magnetic resonance imaging. Oxacillin was administered to cover Staphylococcus aureus, the most common pathogen of pyomyositis. Bilateral hip pain improved within 72 hours after antibiotic usage, but follow-up magnetic resonance imaging after 15 days of treatment revealed well-defined abscess and osteomyelitis of both femoral heads. Abscess incision and drainage were performed, and cultures of the drained pus grew no microorganisms. The patient completed 5 weeks of oxacillin treatment after the operation and recovered with a full range of motion of both hips. There was no residual disability. This is the first report of bilateral hip pain caused by pyomyositis as the initial presentation of CML. Pyomyositis needs to be considered in the differential diagnosis of hip pain in pediatric patients.

Second line BCR/ABK TKI-associated severe adverse events:preferential occurrence in patients with comorbidities

Chronic myeloid leukemia (CML) is a hematopoietic neoplasm characterized by uncontrolled myeloproliferation, basophilia, and the BCR/ABL oncoprotein. The natural course of CML includes a chronic phase, an accelerated phase, and a blast phase that resembles an acute leukemia. The BCR/ABL tyrosine-

Diagnosis of Chronic Myeloid Leukemia Early in Pregnancy

Imatinib therapy has revolutionized the clinical course of Chronic Myeloid Leukemia. The unexpected prolonged survival raised several issues on the quality of life and on the possibility to parent childs.