F-X Mahon

Pharmacokinetic/pharmacodynamic correlation and blood-level testing in imatinib therapy for chronic myeloid leukemia

Imatinib is the current standard of care in the treatment of chronic myeloid leukemia (CML), inducing durable responses and prolonged progression-free survival. However, plasma exposure to the drug from a given dosing regimen can vary widely among patients. Reasons for this may include incomplete adherence, intrinsic variations in the metabolism of imatinib, and drug–drug interactions. Data from two recent studies have shown a correlation between imatinib trough plasma concentration and clinical response, leading to suggestions that maintaining imatinib blood concentrations above ∼1000 ng/ml might be associated with improved outcomes. In patients who do not respond as well as expected to initial imatinib treatment, measurement of trough plasma concentration could assist with decisions about whether to increase the dose. Blood-level testing may also be helpful in other clinical scenarios: for example, when poor adherence is suspected, adverse reactions are unusually severe, or there is a possible drug–drug interaction. Further work is required to confirm prospectively the link between imatinib plasma concentrations and response, and to define effective trough concentrations in different patient populations. However, based on the current data, imatinib blood-level testing seems to be a useful aid when making clinical decisions in CML.

The concept of treatment-free remission in chronic myeloid leukemia

The advent of tyrosine kinase inhibitors (TKI) into the management of patients with chronic myeloid leukemia (CML) has profoundly improved prognosis. Survival of responders is approaching that of the general population but lifelong treatment is still recommended. In several trials, TKI treatment has been stopped successfully in approximately half of the patients with deep molecular response. This has prompted the development of a new concept in the evaluation of CML patients known as ‘treatment-free remission’. The future in CML treatment will be to define criteria for the safe and most promising discontinuation of TKI on one hand, and, on the other, to increase the number of patients available for such an attempt. Until safe criteria have been defined, discontinuation of therapy is still experimental and should be restricted to clinical trials or registries. This review will provide an overview of current knowledge as well as an outlook on future challenges.

Frontline nilotinib in patients with chronic myeloid leukemia in chronic phase: results from the European ENEST1st study

The Evaluating Nilotinib Efficacy and Safety in Clinical Trials as First-Line Treatment (ENEST1st) study included 1089 patients with newly diagnosed chronic myeloid leukemia in chronic phase. The rate of deep molecular response (MR4 (BCR-ABL1⩽0.01% on the International Scale or undetectable BCR-ABL1 with ⩾10 000 ABL1 transcripts)) at 18 months was evaluated as the primary end point, with molecular responses monitored by the European Treatment and Outcome Study network of standardized laboratories. This analysis was conducted after all patients had completed 24 months of study treatment (80.9% of patients) or discontinued early. In patients with typical BCR-ABL1 transcripts and ⩽3 months of prior imatinib therapy, 38.4% (404/1052) achieved MR4 at 18 months. Six patients (0.6%) developed accelerated or blastic phase, and 13 (1.2%) died. The safety profile of nilotinib was consistent with that of previous studies, although the frequencies of some nilotinib-associated adverse events were lower (for example, rash, 21.4%). Ischemic cardiovascular events occurred in 6.0% of patients. Routine monitoring of lipid and glucose levels was not mandated in the protocol. These results support the use of frontline nilotinib, particularly when achievement of a deep molecular response (a prerequisite for attempting treatment-free remission in clinical trials) is a treatment goal.

Increased proportion of mature NK cells is associated with successful imatinib discontinuation in chronic myeloid leukemia

Recent studies suggest that a proportion of chronic myeloid leukemia (CML) patients in deep molecular remission can discontinue the tyrosine kinase inhibitor (TKI) treatment without disease relapse. In this multi-center, prospective clinical trial (EURO-SKI, NCT01596114) we analyzed the function and phenotype of T and NK cells and their relation to successful TKI cessation. Lymphocyte subclasses were measured from 100 imatinib-treated patients at baseline and 1 month after the discontinuation, and functional characterization of NK and T cells was done from 45 patients. The proportion of NK cells was associated with the molecular relapse-free survival as patients with higher than median NK-cell percentage at the time of drug discontinuation had better probability to stay in remission. Similar association was not found with T or B cells or their subsets. In non-relapsing patients the NK-cell phenotype was mature, whereas patients with more naïve CD56bright NK cells had decreased relapse-free survival. In addition, the TNF-α/IFN-γ cytokine secretion by NK cells correlated with the successful drug discontinuation. Our results highlight the role of NK cells in sustaining remission and strengthen the status of CML as an immunogenic tumor warranting novel clinical trials with immunomodulating agents.

Mutation analysis of TET2 , IDH1 , IDH2 and ASXL1 in chronic myeloid leukemia

Acquired somatic mutations affecting TET oncogene family number 2 (TET2), isocitrate dehydrogenase 1 and 2 (IDH1 and IDH2) and additional sex combs-like 1 (ASXL1) have been recently described in BCR–ABL-negative neoplasm, myeloproliferative neoplasms (MPN), myelodysplastic syndrome (MDS), MDS/MPN and acute myeloid leukemias (AMLs), including transformations of MDS or MPN.1 TET2 mutations are diverse (frameshift, nonsense and missense mutations) and do not cluster in a particular region of the protein. In contrast, mutations affecting IDH1/2 and ASXL1 are found in restricted regions.2, 3 None of these gene alterations are disease-specific and their incidence ranges from rare to 40%.1 Another important observation from recent related studies is that these mutations can coexist with other pathogenetically relevant mutations including JAK2V617F, sometimes before or emerging in a subclone.4 Therefore, and especially in view of the occurrence of these mutations across varying molecular profiles, their contribution to disease initiation, clonal evolution or blastic transformation is not clear. Also, the possibility of independently emerging multiple abnormal clones rather than monoclonal myeloproliferation is raised. In chronic myeloid leukemia (CML), BCR–ABL fusion is known to be the driver mutation. However, leukemogenesis is a complex process, and genomic heterogeneity of the chronic phase (CP) of the disease has been reported.5 This intrinsic heterogeneity at the molecular level could support a causative link with the varying response to treatment and disease progression. Furthermore, we still do not know for sure whether the BCR–ABL fusion is really the initiating lesion. In order to describe TET2, IDH1, IDH2 and ASXL1 mutation prevalence, to investigate whether these defects could represent an important event in CML initiation, and to define the relationships between acquisition of these mutations and CML transformation and/or imatinib (IM) resistance, here we report on a mutation analysis done on paired samples from CML at diagnosis (untreated), time of IM response and, when available, complete cytogenetic response (CCR) in 91 CML patients treated with IM as first line—most of them from the French SPIRIT trial—and presenting five profiles of IM response at the time of analysis: (1) 25 had CCR and major molecular response (MMR) at 12 months of IM; (2) 11 were in CCR but showed a Ph negative (Ph−) clonal evolution 12–24 months after IM (median 12 months); (3) 20 were in partial cytogenetic response (4 minor and 16 major), 3 of them with additional cytogenetic abnormalities (−Y, +8, +8) 18 months after IM and referred as primary resistant (R1); (4) 20 were in acute transformation (14 myeloid, 6 lymphoid) 4–72 months after onset of IM (median 12 months); and (5) 15 referred as secondary IM-resistant (R2) had relapse in CP (4 molecular relapse, 6 loss of CCR and 5 hematologic relapse). Sokal scores were homogeneously distributed among the five groups except for group 2, which did not have higher scores. Except for group 1, BCR–ABL residual disease evaluated by RQ-PCR remained high (median value 0.09%, according to the International Scale (IS)).

Rapid analysis and efficient selection of human transduced primitive hematopoietic cells using the humanized S65T green fluorescent protein

We have developed an efficient and rapid method to analyze transduction in human hematopoietic cells and to select them. We constructed two retroviral vectors using the recombinant humanized S65T green fluorescent protein (rHGFP) gene. Transduced cells appeared with specific green fluorescence on microscopy or fluorescence-activated cell sorting (FACS) analysis. The rHGFP gene was placed under the control of two different retroviral promotors (LTR) in the LGSN vector and in the SF-GFP vector. Amphotropic retroviruses were tested on NIH/3T3 fibroblasts or human hematopoietic (K562, TF-1) cell lines. Then CD34+ cells isolated from cord blood were infected three times after a 48-h prestimulation with IL-3, IL-6, SCF or with IL-3, IL-6, SCF, GM-CSF, Flt3-L and TPO. After 6 days of expansion, a similar number of total CD34+-derived cells, CD34+ cells and CFC was obtained in non-transduced and transduced cells, demonstrating the absence of toxicity of the GFP. A transduction up to 46% in total CD34+-derived cells and 21% of CD34+ cells was shown by FACS analysis. These results were confirmed by fluorescence of colonies in methyl-cellulose (up to 36% of CFU-GM and up to 25% of BFU-E). The FACS sorting of GFP+ cells led to 83–100% of GFP-positive colonies after 2 weeks of methyl-cellulose culture. Moreover, a mean gene transfer efficiency of 8% was also demonstrated in long-term culture initiating cells (LTC-IC). This rapid and efficient method represents a substantial improvement to monitor gene transfer and retroviral expression of various vectors in characterized human hematopoietic cells.

Development and evaluation of a secondary reference panel for BCR-ABL1 quantification on the International Scale

Molecular monitoring of chronic myeloid leukemia patients using robust BCR-ABL1 tests standardized to the International Scale (IS) is key to proper disease management, especially when treatment cessation is considered. Most laboratories currently use a time-consuming sample exchange process with reference laboratories for IS calibration. A World Health Organization (WHO) BCR-ABL1 reference panel was developed (MR1–MR4), but access to the material is limited. In this study, we describe the development of the first cell-based secondary reference panel that is traceable to and faithfully replicates the WHO panel, with an additional MR4.5 level. The secondary panel was calibrated to IS using digital PCR with ABL1, BCR and GUSB as reference genes and evaluated by 44 laboratories worldwide. Interestingly, we found that >40% of BCR-ABL1 assays showed signs of inadequate optimization such as poor linearity and suboptimal PCR efficiency. Nonetheless, when optimized sample inputs were used, >60% demonstrated satisfactory IS accuracy, precision and/or MR4.5 sensitivity, and 58% obtained IS conversion factors from the secondary reference concordant with their current values. Correlation analysis indicated no significant alterations in %BCR-ABL1 results caused by different assay configurations. More assays achieved good precision and/or sensitivity than IS accuracy, indicating the need for better IS calibration mechanisms.

Low incidence of peripheral arterial disease in patients receiving dasatinib in clinical trials

The oral tyrosine kinase inhibitors (TKI) dasatinib, imatinib, ponatinib, nilotinib and bosutinib each target BCR-ABL, yet are structurally distinct from one another.1, 2, 3, 4, 5, 6 The selection of BCR-ABL TKI treatment for an individual patient is influenced by factors including any previous treatment, likelihood of benefit, risk of toxicities and the potential for end-organ damage resulting from use, particularly as a long-term therapy is expected in chronic myeloid leukemia in chronic phase (CML-CP). Despite considerable overlap of safety profiles, there are distinct drug-specific adverse events associated with each TKI.2

From in vitro to in vivo: intracellular determination of imatinib and nilotinib may be related with clinical outcome

From in vitro to in vivo: intracellular determination of imatinib and nilotinib may be related with clinical outcome

Proposed diagnostic criteria and classification of basophilic leukemias and related disorders

Basophils form a distinct cell lineage within the hematopoietic cell family. In various myeloid neoplasms, including chronic myeloid leukemia, basophilia is frequently seen. Acute and chronic basophilic leukemias, albeit rare, have also been described. However, no generally accepted criteria and classification of basophilic leukemias have been presented to date. To address this unmet need, a series of Working Conferences and other meetings were organized between March 2015 and March 2016. The current article provides a summary of consensus statements from these meetings, together with proposed criteria to delineate acute basophilic leukemia (ABL) from chronic basophilic leukemia (CBL) and primary forms of the disease where no preceding myeloid malignancy is detected, from the more common ‘secondary’ variants. Moreover, the term hyperbasophilia (HB) is proposed for cases with a persistent peripheral basophil count ⩾1000 per μl of blood. This condition, HB, is highly indicative of the presence of an underlying myeloid neoplasm. Therefore, HB is an important checkpoint in the diagnostic algorithm and requires a detailed hematologic investigation. In these patients, an underlying myeloid malignancy is often found and is then labeled with the appendix -baso, whereas primary cases of ABL or CBL are very rare. The criteria and classification proposed in this article should facilitate the diagnosis and management of patients with unexplained basophilia and basophil neoplasms in routine practice, and in clinical studies.