Idiopathic erythrocytosis: a germline disease?

Polycythemia Vera (PV) is typically caused by V617F or exon 12 JAK2 mutations. Little is known about Polycythemia cases where no JAK2 variants can be detected, and no other causes identified. This condition is defined as idiopathic erythrocytosis (IE). We evaluated clinical-laboratory parameters of a cohort of 56 IE patients and we determined their molecular profile at diagnosis with paired blood/buccal-DNA exome-sequencing coupled with a high-depth targeted OncoPanel to identify a possible underling germline or somatic cause. We demonstrated that most of our cohort (40/56: 71.4%) showed no evidence of clonal hematopoiesis, suggesting that IE is, in large part, a germline disorder. We identified 20 low mutation burden somatic variants (Variant allelic fraction, VAF, < 10%) in only 14 (25%) patients, principally involving DNMT3A and TET2. Only 2 patients presented high mutation burden somatic variants, involving DNMT3A, TET2, ASXL1 and WT1. We identified recurrent germline variants in 42 (75%) patients occurring mainly in JAK/STAT, Hypoxia and Iron metabolism pathways, among them: JAK3-V722I and HIF1A-P582S; a high fraction of patients (48.2%) resulted also mutated in homeostatic iron regulatory gene HFE-H63D or C282Y. By generating cellular models, we showed that JAK3-V722I causes activation of the JAK-STAT5 axis and upregulation of EPAS1/HIF2A, while HIF1A-P582S causes suppression of hepcidin mRNA synthesis, suggesting a major role for these variants in the onset of IE.

Practical management of toxicities associated with bosutinib in patients with Philadelphia chromosome-positive chronic myeloid leukemia.

Bosutinib (SKI-606) is an oral, dual Src/Abl tyrosine kinase inhibitor (TKI) approved for treatment of patients with Philadelphia chromosome-positive (Ph+) chronic myeloid leukemia (CML) that is resistant or intolerant to prior TKI therapy or for whom other TKIs are not appropriate choices. The objective of this review is to provide a longitudinal summary of toxicities that may arise during treatment with second-line or later bosutinib in patients with Ph+ chronic phase CML and to provide strategies for managing these toxicities. As bosutinib is not currently indicated for newly diagnosed CML, toxicities associated with first-line treatment are not reviewed. Recognition and optimal management of these toxicities can facilitate patient compliance and affect treatment outcomes.

Treatment Efficacy and Resistance Mechanisms Using the Second-Generation ALK Inhibitor AP26113 in Human NPM-ALK-Positive Anaplastic Large Cell Lymphoma.

UNLABELLED: ALK is a tyrosine kinase receptor involved in a broad range of solid and hematologic tumors. Among 70% to 80% of ALK(+) anaplastic large cell lymphomas (ALCL) are caused by the aberrant oncogenic fusion protein NPM-ALK. Crizotinib was the first clinically relevant ALK inhibitor, now approved for the treatment of late-stage and metastatic cases of lung cancer. However, patients frequently develop drug resistance to Crizotinib, mainly due to the appearance of point mutations located in the ALK kinase domain. Fortunately, other inhibitors are available and in clinical trial, suggesting the potential for second-line therapies to overcome Crizotinib resistance. This study focuses on the ongoing phase I/II trial small-molecule tyrosine kinase inhibitor (TKI) AP26113, by Ariad Pharmaceuticals, which targets both ALK and EGFR. Two NPM-ALK(+) human cell lines, KARPAS-299 and SUP-M2, were grown in the presence of increasing concentrations of AP26113, and eight lines were selected that demonstrated resistance. All lines show IC50 values higher (130 to 1,000-fold) than the parental line. Mechanistically, KARPAS-299 populations resistant to AP26113 show NPM-ALK overexpression, whereas SUP-M2-resistant cells harbor several point mutations spanning the entire ALK kinase domain. In particular, amino acid substitutions: L1196M, S1206C, the double F1174V+L1198F and L1122V+L1196M mutations were identified. The knowledge of the possible appearance of new clinically relevant mechanisms of drug resistance is a useful tool for the management of new TKI-resistant cases. IMPLICATIONS: This work defines reliable ALCL model systems of AP26113 resistance and provides a valuable tool in the management of all cases of relapse upon NPM-ALK-targeted therapy.

A phase 2 trial of ponatinib in Philadelphia chromosome-positive leukemias.

BACKGROUND: Ponatinib is a potent oral tyrosine kinase inhibitor of unmutated and mutated BCR-ABL, including BCR-ABL with the tyrosine kinase inhibitor-refractory threonine-to-isoleucine mutation at position 315 (T315I). We conducted a phase 2 trial of ponatinib in patients with chronic myeloid leukemia (CML) or Philadelphia chromosome-positive acute lymphoblastic leukemia (Ph-positive ALL). METHODS: We enrolled 449 heavily pretreated patients who had CML or Ph-positive ALL with resistance to or unacceptable side effects from dasatinib or nilotinib or who had the BCR-ABL T315I mutation. Ponatinib was administered at an initial dose of 45 mg once daily. The median follow-up was 15 months. RESULTS: Among 267 patients with chronic-phase CML, 56% had a major cytogenetic response (51% of patients with resistance to or unacceptable side effects from dasatinib or nilotinib and 70% of patients with the T315I mutation), 46% had a complete cytogenetic response (40% and 66% in the two subgroups, respectively), and 34% had a major molecular response (27% and 56% in the two subgroups, respectively). Responses were observed regardless of the baseline BCR-ABL kinase domain mutation status and were durable; the estimated rate of a sustained major cytogenetic response of at least 12 months was 91%. No single BCR-ABL mutation conferring resistance to ponatinib was detected. Among 83 patients with accelerated-phase CML, 55% had a major hematologic response and 39% had a major cytogenetic response. Among 62 patients with blast-phase CML, 31% had a major hematologic response and 23% had a major cytogenetic response. Among 32 patients with Ph-positive ALL, 41% had a major hematologic response and 47% had a major cytogenetic response. Common adverse events were thrombocytopenia (in 37% of patients), rash (in 34%), dry skin (in 32%), and abdominal pain (in 22%). Serious arterial thrombotic events were observed in 9% of patients; these events were considered to be treatment-related in 3%. A total of 12% of patients discontinued treatment because of an adverse event. CONCLUSIONS: Ponatinib had significant antileukemic activity across categories of disease stage and mutation status. (Funded by Ariad Pharmaceuticals and others; PACE ClinicalTrials.gov number, NCT01207440 .).

SETBP1 mutations occur in 9% of MDS/MPN and in 4% of MPN cases and are strongly associated with atypical CML, monosomy 7, isochromosome i(17)(q10), ASXL1 and CBL mutations.

Chronic myeloid malignancies are categorized to the three main categories myeloproliferative neoplasms (MPNs), myelodysplastic syndromes (MDSs) and MDS/MPN overlap. So far, no specific genetic alteration profiles have been identified in the MDS/MPN overlap category. Recent studies identified mutations in SET-binding protein 1 (SETBP1) as novel marker in myeloid malignancies, especially in atypical chronic myeloid leukemia (aCML) and related diseases. We analyzed SETBP1 in 1 130 patients with MPN and MDS/MPN overlap and found mutation frequencies of 3.8% and 9.4%, respectively. In particular, there was a high frequency of SETBP1 mutation in aCML (19/60; 31.7%) and MDS/MPN unclassifiable (MDS/MPN, U; 20/240; 9.3%). SETBP1 mutated (SETBP1mut) patients showed significantly higher white blood cell counts and lower platelet counts and hemoglobin levels than SETBP1 wild-type patients. Cytomorphologic evaluation revealed a more dysplastic phenotype in SETBP1mut cases as compared with wild-type cases. We confirm a significant association of SETBP1mut with -7 and isochromosome i(17)(q10). Moreover, SETBP1mut were strongly associated with ASXL1 and CBL mutations (P<0.001 for both) and were mutually exclusive of JAK2 and TET2 mutations. In conclusion, SETBP1mut add an important new diagnostic marker for MDS/MPN and in particular for aCML.

Somatic SF3B1 mutation in myelodysplasia with ring sideroblasts.

BACKGROUND: Myelodysplastic syndromes are a diverse and common group of chronic hematologic cancers. The identification of new genetic lesions could facilitate new diagnostic and therapeutic strategies. METHODS: We used massively parallel sequencing technology to identify somatically acquired point mutations across all protein-coding exons in the genome in 9 patients with low-grade myelodysplasia. Targeted resequencing of the gene encoding RNA splicing factor 3B, subunit 1 (SF3B1), was also performed in a cohort of 2087 patients with myeloid or other cancers. RESULTS: We identified 64 point mutations in the 9 patients. Recurrent somatically acquired mutations were identified in SF3B1. Follow-up revealed SF3B1 mutations in 72 of 354 patients (20%) with myelodysplastic syndromes, with particularly high frequency among patients whose disease was characterized by ring sideroblasts (53 of 82 [65%]). The gene was also mutated in 1 to 5% of patients with a variety of other tumor types. The observed mutations were less deleterious than was expected on the basis of chance, suggesting that the mutated protein retains structural integrity with altered function. SF3B1 mutations were associated with down-regulation of key gene networks, including core mitochondrial pathways. Clinically, patients with SF3B1 mutations had fewer cytopenias and longer event-free survival than patients without SF3B1 mutations. CONCLUSIONS: Mutations in SF3B1 implicate abnormalities of messenger RNA splicing in the pathogenesis of myelodysplastic syndromes. (Funded by the Wellcome Trust and others.).

BCR and BCR-ABL regulation during myeloid differentiation in healthy donors and in chronic phase/blast crisis CML patients.

Chronic myeloid leukemia (CML) is caused by the BCR-ABL hybrid gene. The molecular mechanisms leading from chronic phase (CP) to blast crisis (BC) are not understood. However, both the presence and the levels of BCR-ABL seem to be important for CML progression. BCR-ABL is under the transcriptional control of BCR promoter. Here we focused on the gene expression control of BCR and BCR-ABL upon myeloid differentiation in healthy donors (HDs), CP and BC patients. As previously reported, BCR-ABL is downregulated during myeloid maturation in CP patients. A similar pattern was detected for BCR (but not for ABL) in CP-CML and in HD, thus suggesting that the two genes may be under a similar transcriptional control. In BC this mechanism is similarly impaired for both BCR-ABL and BCR. These data indicate the presence of an 'in trans' deregulated transcription of both BCR and BCR-ABL promoters, associated with CML progression.

Efficacy and safety of dasatinib in imatinib-resistant or -intolerant patients with chronic myeloid leukemia in blast phase.

Dasatinib is an inhibitor of BCR-ABL and SRC-family kinases for patients with imatinib-resistant or -intolerant chronic myelogenous leukemia (CML). In this international phase II trial, dasatinib was administered orally (70 mg twice daily) to patients with myeloid blast phase (MBP, n=109) or lymphoid blast phase (LBP, n=48) CML. After a minimum follow-up of 12 months (range 0.03-20.7 months), major hematologic responses were induced in 34% (MBP-CML) and 35% (LBP-CML) of patients. Major cytogenetic responses were attained in 33% (MBP-CML) and 52% (LBP-CML) of patients and complete cytogenetic responses were attained in 26 and 46%, respectively. Median progression-free survival was 6.7 (MBP-CML) and 3.0 (LBP-CML) months. Median overall survival was 11.8 (MBP-CML) and 5.3 (LBP-CML) months. Overall, dasatinib had acceptable tolerability. Fluid retention events were more frequent in the MBP-CML than the LBP-CML cohort: pleural effusion occurred in 36 and 13% (all grades) and 15 and 6% (grades 3/4), respectively. Other non-hematologic side effects were primarily grade 1/2; grade 3/4 events were recorded in

Anaplastic lymphoma kinase and its signalling molecules as novel targets in lymphoma therapy.

A crucial issue in the development of molecularly-targeted anticancer therapies is the identification of appropriate molecules whose targeting would result in tumour regression with a minimal level of systemic toxicity. Anaplastic lymphoma kinase (ALK) is a transmembrane receptor tyrosine kinase, normally expressed at low levels in the nervous system. As a consequence of chromosomal translocations involving the alk gene (2p23), ALK is also aberrantly expressed and constitutively activated in approximately 60% of CD30+ anaplastic large cell lymphomas (ALCLs). Due to the selective overexpression of ALK in tumour cells, its direct involvement in the process of malignant transformation and its frequent expression in ALCL patients, the authors recognise ALK as a suitable candidate for the development of molecularly targeted strategies for the therapeutic treatment of ALK-positive lymphomas. Strategies targeting ALK directly or indirectly via the inhibition of the protein networks responsible for ALK oncogenic signalling are discussed.

Effect of imatinib on haematopoietic recovery following idarubicin exposure.

SCF is a potent pro-proliferative cytokine crucial for haematopoiesis, which binds to c-kit and activates its tyrosine kinase activity. Inactivating mutations of either SCF or c-kit have been described in mice and lead to increased sensitivity to treatment with ionising radiation. Imatinib is a tyrosine kinase inhibitor with high affinity for c-Abl, PDGFR and c-kit. In this study we investigated the effect of concomitant administration of imatinib and idarubicin, an anthracycline with haematosuppressive activity, in nu/nu mice and murine bone marrow cells. Double-treated animals showed significantly increased mortality compared to mice that received imatinib or idarubicin alone only when idarubicin and imatinib were given simultaneously. The combined treatment induced a more severe neutropenia with a slower recovery when compared to mice treated with idarubicin alone. The myeloid metaplasia usually observed in the spleen after idarubicin treatment was absent in mice co-treated with imatinib. Bone marrow from double-treated animals also showed decreased numbers of megakaryocytes and myeloid precursor cells. In vitro culture of murine bone marrow cells in the presence of imatinib inhibited SCF-induced proliferation and recovery from treatment with idarubicin. Our results indicate that the simultaneous administration of imatinib enhances idarubicin-induced haematopoietic toxicity in vivo and in vitro.

Inhibition of promyelocytic leukemia (PML)/retinoic acid receptor-alpha and PML expression in acute promyelocytic leukemia cells by anti-PML peptide nucleic acid.

The fusion protein promyelocytic leukemia (PML)/retinoic acid receptor (RAR)alpha is tightly linked to the pathogenesis of acute promyelocytic leukemia (APL); hence, it represents a tumor-associated, transformation-related molecule. In this study, three anti-PML adamantyl-conjugated peptide nucleic acid (PNA) oligomers previously described as in vitro inhibitors of PML/RARalpha translation were combined and used to block PML/RARalpha synthesis in NB4 cells. Cationic liposomes were used to achieve sufficient delivery of PNAs into the cells. Upon treatment of cells with the liposome/PNA mixture, enhanced cellular uptake of PNA (approximately 5-fold compared with control) was obtained. Concomitantly, a substantial reduction (>90%) of the expression of PML/RARalpha was observed when all of the three PNAs were used together. This resulted in a dramatic effect on the number and viability of NB4 cells in culture after 48 h of treatment. This phenomenon was preceded by induction of apoptosis that could be observed 24 h after treatment. No sign of granulocytic differentiation was observed after treatment. These effects were also noted on other leukemic cell lines that express PML but not the fusion transcript. These results show that it is possible to deliver PNA into hematopoietic cells and obtain specific gene inhibition, and they suggest that a growth inhibitory effect on acute promyelocytic leukemia cells can be obtained through the block of PML/RARalpha and PML expression.

Sensitivity to the abl inhibitor STI571 in fresh leukaemic cells obtained from chronic myelogenous leukaemia patients in different stages of disease.

STI571 (CGP57148B) is an inhibitor of BCR/ABL, the cause of chronic myeloid leukaemia (CML). A difference exists between CML patients in chronic phase, in which responses to STI571are durable, and patients in blast crisis, who generally experience only transient responses. Leukaemic cells from six CML patients from whom samples could be obtained during chronic phase and at the time of blast crisis (BC) were compared for sensitivity to STI571, using an in vitro assay. BC samples showed a sensitivity similar to that obtained during chronic phase, suggesting that no substantial intrinsic resistance to STI571 was present in BC.