The Bruton's tyrosine kinase (BTK) inhibitor ibrutinib is inducing durable responses in chronic lymphocytic leukemia (CLL) patients with refractory/relapsed disease or with TP53 defect, with BTK and phospholipase C gamma 2 (PLCG2) mutations representing the predominant mechanisms conferring secondary ibrutinib resistance. To understand the landscape of genomic changes and the dynamics of subclonal architecture associated with ibrutinib treatment, an ultra‐deep next‐generation sequencing analysis of 30 recurrently mutated genes was performed on sequential samples of 20 patients, collected before and during single‐agent ibrutinib treatment. Mutations in the SF3B1, MGAand BIRC3 genes were enriched during ibrutinib treatment, while aberrations in the BTK, PLCG2, RIPK1, NFKBIE and XPO1 genes were exclusively detected in posttreatment samples. Besides the canonical mutations, four novel BTK mutations and three previously unreported PLCG2 variants were identified. BTK and PLCG2 mutations were backtracked in five patients using digital droplet PCR and were detectable on average 10.5 months before clinical relapse. With a median follow‐up time of 36.5 months, 7/9 patients harboring BTK mutations showed disease progression based on clinical and/or laboratory features. In conclusion, subclonal heterogeneity, dynamic clonal selection and various patterns of clonal variegation were identified with novel resistance‐associated BTK mutations in individual patients treated with ibrutinib.

References

Background: Additional chromosome abnormalities (ACAs), mutations of the BCR-ABL tyrosine kinase domain (TKD) and BCR-ABL splice variants may cause resistance to first- and second-generation tyrosine kinase inhibitors (TKIs) in chronic myelogenous leukemia (CML) and Philadelphia-positive (Ph+) acute lymphoid leukemia (ALL). Methods: Karyotyping and BCR-ABL TKD mutation screening were performed in 71 imatinib-resistant CML patients and 6 Ph+ ALL patients. A total of 56 out of these 77 patients received second-generation TKI. Results: ACAs were present in 30 of 65 imatinib-resistant patients (46%). In 27 of 74 imatinib-resistant patients (36%), 15 different BCR-ABL TKD mutations were detected. Mutations were found in 25% of chronic-phase patients (12/47), 33% of accelerated-phase patients (5/15), 71% of blast crisis CML patients (5/7) and 100% of ALL patients. In nilotinib-resistant patients, Y253H, T315I and F359I/V mutations were detected; in dasatinib-resistant patients, L248M, E279K and T315I mutations were detected. T315I was found more frequently in patients on dasatinib than on imatinib therapy. The presence of ACAs predicted shorter survival during first- and second-generation TKI therapy, while TKD mutations only influenced survival during second-generation TKI therapy. Conclusion: For patients with TKI resistance, mutation and ACA screening may play a role in identifying patients with poorer prognosis.

Mutations of isocitrate dehydrogenase 1 and 2 (IDH1/2) are genetic alterations in acute myeloid leukemia (AML). The aim of our study was to investigate the frequency and prognostic effect of IDH1/2 mutations together followed by an individual analysis of each substitution in a Hungarian cohort consisting of 376 patients with AML. IDH1(mut) and IDH2(mut) were mutually exclusive, detected in 8.5% and 7.5% of cases, respectively. IDH1/2(mut) was associated with: older age (p = 0.001), higher average platelet count (p = 0.001), intermediate karyotype (p < 0.0001), NPM1(mut) (p = 0.022) and lower mRNA expression level of ABCG2 gene (p = 0.006). Overall survival (OS), remission and relapse rates were not different in IDH1(mut) or IDH2(mut) vs. IDH(neg). IDH1(mut) and IDH2(mut) were associated differently with NPM1(mut); co-occurrence was observed in 14.3% of IDH1 R132C vs. 70% of R132H carriers (p = 0.02) and in 47.4% of IDH2 R140Q vs. 0% of R172K carriers (p = 0.02). IDH1 R132H negatively influenced OS compared to IDH(neg) (p = 0.02) or R132C (p = 0.019). Particular amino acid changes affecting the same IDH1 codon influence the clinical characteristics and treatment outcome in AML.

The online version of this article has a Supplementary Appendix. BackgroundPrognostic risk stratification according to acquired or inherited genetic alterations has received increasing attention in acute myeloid leukemia in recent years. A germline Janus kinase 2 haplotype designated as the 46/1 haplotype has been reported to be associated with an inherited predisposition to myeloproliferative neoplasms, and also to acute myeloid leukemia with normal karyotype. The aim of this study was to assess the prognostic impact of the 46/1 haplotype on disease characteristics and treatment outcome in acute myeloid leukemia. Design and MethodsJanus kinase 2 rs12343867 single nucleotide polymorphism tagging the 46/1 haplotype was genotyped by LightCycler technology applying melting curve analysis with the hybridization probe detection format in 176 patients with acute myeloid leukemia under 60 years diagnosed consecutively and treated with curative intent. ResultsThe morphological subtype of acute myeloid leukemia with maturation was less frequent among 46/1 carriers than among non-carriers (5.6% versus 17.2%, P=0.018, cytogenetically normal subgroup: 4.3% versus 20.6%, P=0.031), while the morphological distribution shifted towards the myelomonocytoid form in 46/1 haplotype carriers (28.1% versus 14.9%, P=0.044, cytogenetically normal subgroup: 34.0% versus 11.8%, P=0.035). In cytogenetically normal cases of acute myeloid leukemia, the 46/1 carriers had a considerably lower remission rate (78.7% versus 94.1%, P=0.064) and more deaths in remission or in aplasia caused by infections (46.8% versus 23.5%, P=0.038), resulting in the 46/1 carriers having shorter disease-free survival and overall survival compared to the 46/1 non-carriers. In multivariate analysis, the 46/1 haplotype was an independent adverse prognostic factor for disease-free survival (P=0.024) and overall survival (P=0.024) in patients with a normal karyotype. Janus kinase 2 46/1 haplotype had no impact on prognosis in the subgroup with abnormal karyotype. ConclusionsJanus kinase 2 46/1 haplotype influences morphological distribution, increasing the predisposition towards an acute myelomonocytoid form. It may be a novel, independent unfavorable risk factor in acute myeloid leukemia with a normal karyotype.Key words: acute myeloid leukemia, prognosis, Janus kinase 2, 46/1 haplotype. Haematologica 2011;96(11):1613-1618. doi:10.3324/haematol.2011 This is an open-access paper. Citation: Nahajevszky S, Andrikovics H, Batai A, Adam E, BorsThe prognostic impact of germline 46/1 haplotype of Janus kinase 2 in cytogenetically normal acute myeloid leukemia

There is an increasing body of evidence that suggests that genes involved in cell fate decisions and pattern formation during development also play a key role in the continuous cell fate decisions made by adult tissue stem cells. Here we show that prolonged in vitro culture (14 days) of murine bone marrow lineage negative cells in medium supplemented with three early acting cytokines (stem cell factor, Flk-2/Flt-3 ligand, thrombopoietin) and with immobilized Notch ligand, Jagged-1, resulted in robust expansion of serially transplantable hematopoietic stem cells with long-term repopulating ability. We found that the absolute number of marrow cells was increased approximately 8 to 14-fold in all cultures containing recombinant growth factors. However, the frequency of high quality stem cells was markedly reduced at the same time, except in cultures containing growth factors and Jagged-1-coated Sepharose-4B beads. The absolute number of hematopoietic cells with long-term repopulating ability was increased approximately 10 to 20-fold in the presence of multivalent Notch ligand. These results support a role for combinatorial effects by Notch and cytokine-induced signaling pathways in regulating hematopoietic stem cell fate and to a potential role for Notch ligand in increasing cell numbers in clinical stem cell transplantation.

The chimeric bcr-abl gene encodes a constitutively active tyrosine kinase that leads to abnormal transduction of growth and survival signals leading to chronic myeloid leukemia (CML). According to our previous observations, in vitro differentiation of several erythroid cell lines is accompanied by the downregulation of extracellular signal-regulated kinases (ERK)1/2 mitogen-activated protein kinase (MAPK) activities. In this work we investigated whether ERKs have a decisive role in either the erythroid differentiation process or apoptosis of bcr-abl+ K562 cells by means of direct (MEK1/2 inhibitor UO126) and indirect (reduced Bcr-Abl function) inhibition of their activities. We found that both Gleevec and UO126 induced hemoglobin expression. Gleevec treatment reduced the phosphorylation of Bcr-Abl, ERK and STAT-5 for up to 24 h, decreased Bcl-XL levels, and induced caspase-3-dependent apoptosis. In contrast, UO126 treatment resulted in only a transient decrease of ERK activity and did not induce cell death. For studying the effect of reduced Bcr-Abl function on erythroid differentiation at the level of the bcr-abl transcript, we applied the siRNA approach. Stable degradation of bcr-abl mRNA was achieved by using a retroviral vector with enhanced green fluorescent protein (EGFP) reporter. Despite a high (>90%) transduction efficiency we detected only a transient decrease in Bcr-Abl protein and in phosphorylated ERK1/2 levels. This transient change in Bcr-Abl signaling was sufficient to induce hemoglobin expression without significant cell death. These results suggest that by transiently reducing Bcr-Abl function it is possible to overcome the differentiation blockade without evoking apoptosis in CML cells and that reduced ERK activity may have a crucial role in this process.

During the formation of the Philadelphia (Ph) chromosome, in the majority of chronic myelogenous leukemia (CML) patients, the chromosome 22 breakpoint is located in the major breakpoint cluster region of the BCR gene (M-bcr). Minor and micro breakpoint cluster regions (m-bcr with e1a2 transcript and micro-bcr with e19a2 transcript) are rarely affected and have been suggested to be associated with peculiar CML phenotypes. Despite the different clinical characteristics, it is currently not established, whether different therapeutic options are preferably recommended for the treatment of e1a2 or e19a2 CML. Here we report two patients with e1a2 and one patient with e19a2 translocations, treated with different approaches including imatinib. First and second line imatinib treatments induced haematologic response in all of the three patients, and major cytogenetic response in one patient with e1a2, as well as in the patient with e19a2 CML. However, relapse occurred in the patient with e19a2 CML, possibly caused by the presence of additional chromosomal abnormalities such as an extra Ph chromosome, and loss of chromosome Y. Stem cell transplantation (SCT) therapy caused complete haematologic response with molecular remission; however, the patient died of infectious complication. We conclude that in patients with rare BCR-ABL variants, the effectiveness of imatininb treatment may be influenced by the CML stage besides the actual molecular type of the rare transcript. However, this conclusion cannot be generalized to larger patient groups with rare BCR-ABL variants for which further studies may be needed.