BCR-ABL kinase domain mutation analysis in chronic myeloid leukemia patients treated with tyrosine kinase inhibitors: recommendations from an expert panel on behalf of European LeukemiaNet

Mutations in the Bcr-Abl kinase domain may cause, or contribute to, resistance to tyrosine kinase inhibitors (TKIs) in chronic myeloid leukemia patients. Recommendations aimed to rationalize the use of BCR-ABL mutation testing in chronic myeloid leukemia have been compiled by a panel of experts appointed by the European LeukemiaNet (ELN) and European Treatment and Outcome Study and are here reported. Based on a critical review of the literature and, whenever necessary, on panelists' experience, key issues were identified and discussed concerning: (1) when to perform mutation analysis, (2) how to perform it, and (3) how to translate results into clinical practice. In chronic phase patients receiving imatinib first-line, mutation analysis is recommended only in case of failure or suboptimal response according to the ELN criteria. In imatinib-resistant patients receiving an alternative TKI, mutation analysis is recommended in case of hematologic or cytogenetic failure as provisionally defined by the ELN. The recommended methodology is direct sequencing, although it may be preceded by screening with other techniques, such as denaturing-high performance liquid chromatography. In all the cases outlined within this abstract, a positive result is an indication for therapeutic change. Some specific mutations weigh on TKI selection.     

Novel imatinib-sensitive PDGFRA-activating point mutations in hypereosinophilic syndrome induce growth factor independence and leukemia-like disease

The FIP1L1-PDGFRA fusion is seen in a fraction of cases with a presumptive diagnosis of hypereosinophilic syndrome (HES). However, because most HES patients lack FIP1L1-PDGFRA, we studied whether they harbor activating mutations of the PDGFRA gene. Sequencing of 87 FIP1L1-PDGFRA-negative HES patients revealed several novel PDGFRA point mutations (R481G, L507P, I562M, H570R, H650Q, N659S, L705P, R748G, and Y849S). When cloned into 32D cells, N659S and Y849S and-on selection for high expressors-also H650Q and R748G mutants induced growth factor-independent proliferation, clonogenic growth, and constitutive phosphorylation of PDGFRA and Stat5. Imatinib antagonized Stat5 phosphorylation. Mutations involving positions 659 and 849 had been shown previously to possess transforming potential in gastrointestinal stromal tumors. Because H650Q and R748G mutants possessed only weak transforming activity, we injected 32D cells harboring these mutants or FIP1L1-PDGFRA into mice and found that they induced a leukemia-like disease. Oral imatinib treatment significantly decreased leukemic growth in vivo and prolonged survival. In conclusion, our data provide evidence that imatinib-sensitive PDGFRA point mutations play an important role in the pathogenesis of HES and we propose that more research should be performed to further define the frequency and treatment response of PDGFRA mutations in FIP1L1-PDGFRA-negative HES patients.     

Nilotinib is effective in patients with chronic myeloid leukemia in chronic phase after imatinib resistance or intolerance: 24-month follow-up results

Nilotinib is a potent selective inhibitor of the BCR-ABL tyrosine kinase approved for use in patients with newly diagnosed chronic myeloid leukemia in chronic phase (CML-CP), and in CML-CP and CML-accelerated phase after imatinib failure. Nilotinib (400 mg twice daily) was approved on the basis of the initial results of this phase 2 open-label study. The primary study endpoint was the proportion of patients achieving major cytogenetic response (CyR). All patients were followed for ≥ 24 months or discontinued early. Of 321 patients, 124 (39%) continue on nilotinib treatment. Overall, 59% of patients achieved major CyR; this was complete CyR (CCyR) in 44%. Of patients achieving CCyR, 56% achieved major molecular response. CyRs were durable, with 84% of patients who achieved CCyR maintaining response at 24 months. The overall survival at 24 months was 87%. Adverse events were mostly mild to moderate, generally transient, and easily managed. This study indicates that nilotinib is effective, with a manageable safety profile, and can provide favorable long-term benefits for patients with CML-CP after imatinib failure.     

A co-operative evaluation of different methods of detecting BCR-ABL kinase domain mutations in patients with chronic myeloid leukemia on second-line dasatinib or nilotinib therapy after failure of imatinib

Background

Various techniques have been employed to detect BCR-ABL kinase domain mutations in patients with chronic myeloid leukemia who are resistant to imatinib. This has led to different reported frequencies of mutations and the finding of a heterogeneous pattern of individual mutations.

Design and Methods

We compared direct sequencing alone and in combination with denaturing high-performance liquid chromatography and two high-sensitivity allele-specific oligonucleotide polymerase chain reaction approaches for analysis of BCR-ABL mutations in 200 blinded cDNA samples prior to and during second-line dasatinib or nilotinib therapy in patients with chronic myeloid leukemia in whom imatinib treatment had failed.

Results

One hundred and fourteen mutations were detected by both direct sequencing alone or in combination with high performance liquid chromatography and 13 mutations were additionally detected by the combined technique. Eighty of 83 mutations (96%) within a selected panel of 11 key mutations were confirmed by both allele-specific oligonucleotide polymerase chain reaction techniques and 62 mutations were identified in addition to those detected by combined liquid chromatography and direct sequencing, indicating the presence and a high prevalence of low-level mutations in this cohort of patients. Furthermore, 125 mutations were detected by only one allele-specific oligonucleotide polymerase chain reaction technique. Pre-existing mutations were traceable 4.5 months longer and emerging clones were detectable 3.0 months earlier by allele-specific oligonucleotide polymerase chain reaction than by direct sequencing together with liquid chromatography.

Conclusions

Our results suggest that denaturing high performance liquid chromatography combined with direct sequencing is a reliable screening technique for the detection of BCR-ABL kinase domain mutations. Allele-specific oligonucleotide polymerase chain reaction further increases the number of detected mutations and indicates a high prevalence of mutations at a low level. The clinical impact of such low-level mutations remains uncertain and requires further investigation. Allele-specific oligonucleotide polymerase chain reaction allows detection of defined mutations at a lower level than does denaturing high performance liquid chromatography combined with direct sequencing and may, therefore, provide clinical benefit by permitting early reconsideration of therapeutic strategies.     

Transient response to imatinib in a chronic eosinophilic leukemia associated with ins(9;4)(q33;q12q25) and a CDK5RAP2-PDGFRA fusion gene

Chronic myeloproliferative disorders with rearrangements of the platelet-derived growth factor receptor A (PDGFRA) gene at chromosome band 4q12 have shown excellent responses to targeted therapy with imatinib. Here we report a female patient who presented with advanced phase of a chronic eosinophilic leukemia. Cytogenetic analysis revealed an ins(9;4)(q33;q12q25) in 5 of 21 metaphases. FISH analysis with flanking BAC probes indicated that PDGFRA was disrupted. A novel mRNA in-frame fusion between exon 13 of the CDK5 regulatory subunit associated protein 2 (CDK5RAP2) gene, a 40-bp insert that was partially derived from an inverted sequence stretch of PDGFRA intron 9, and a truncated PDGFRA exon 12 was identified by 5'-RACE-PCR. CDK5RAP2 encodes a protein that is believed to be involved in centrosomal regulation. The predicted CDK5RAP2-PDGFRA protein consists of 1,003 amino acids and retains both tyrosine kinase domains of PDGFRA and several potential dimerization domains of CDK5RAP2. Despite achieving complete cytogenetic and molecular remission on imatinib, the patient relapsed with imatinib-resistant acute myeloid leukemia that was characterized by a normal karyotype, absence of detectable CDK5RAP2-PDGFRA mRNA, and a newly acquired G12D NRAS mutation.     

Continuous complete hematological and cytogenetic remission with molecular minimal residual disease 9 years after discontinuation of interferon-alpha in a patient with Philadelphia chromosome-positive chronic myeloid leukemia

Interferon-alpha has been used as standard therapy for patients with Philadelphia-positive chronic myeloid leukemia (CML) for more than 20 years. Recently randomized trials have shown a superiority of the tyrosine kinase inhibitor imatinib in respect to its efficacy to induce complete hematological and cytogenetic remissions and more importantly in overall survival. Although follow-up is much shorter for imatinib than for interferon-alpha, this data changed the treatment algorithms in this disease. At the end of the era of interferon-alpha as a single-drug first-line treatment for most patients we present a case report which exemplifies a rare but exciting property of interferon-alpha in CML: the induction of complete hematological and cytogenetic remissions which can persist over years after discontinuation of the drug. Hence, the enrollment of CML patients in clinical trials which explore a combination treatment of imatinib and interferon-alpha is warranted.     

Impact of NOD2 polymorphisms on infectious complications following chemotherapy in patients with acute myeloid leukaemia

We sought to investigate the relationship between polymorphisms of the NOD2 gene and infectious complications following intensive induction chemotherapy in patients with acute myeloid leukaemia (AML). We hypothesised that single nucleotide polymorphisms (SNPs) of the NOD2 gene are associated with a higher rate of infections during the phase of severe neutropenia. In 131 AML patients receiving induction therapy, the presence of the three most frequent polymorphisms of NOD2 (Arg702Trp, Gly908Arg, Leu1007fsinsC) was analysed. SNP analyses by means of genomic PCR incorporating fluorescence-labelled probes with characteristic melting curves were performed using the LightCycler platform. Our data suggest a significantly lower probability of mucositis or enteritis in AML patients lacking any of the three evaluated NOD2 polymorphisms. Furthermore, bloodstream cultures of AML patients carrying either a missense or a frameshift mutation of NOD2 were significantly more frequently tested positive concerning Streptococcus spp. In contrast, the presence of NOD2 polymorphisms had no impact on such important infectious complications as systemic inflammatory response syndrome or sepsis, the rate of central venous catheter infections or the incidence of pneumonia including fungal infections. Our data represent one of the first reports investigating the impact of polymorphisms of the innate immune system on infectious complications in patients with neutropenia following chemotherapy. A correlation between NOD2 polymorphisms and infectious events in AML patients is demonstrated.     

A Stability Analysis of a Modified Version of the Chi-Square Ratio Statistic: Implications for Equivalence Testing of Aerodynamic Particle Size Distribution

Demonstration of equivalence in aerodynamic particle size distribution (APSD; e.g., by comparing cascade impactor (CI) profiles) constitutes one of key in vitro tests for supporting bioequivalence between test (T) and reference (R) orally inhaled drug products (OIDPs). A chi-square ratio statistic (CSRS) was previously proposed for equivalence testing of CI profiles. However, it was reported that the CSRS could not consistently discriminate between equivalent and inequivalent CI profiles. The objective of the overall project was to develop a robust and sensitive methodology for assessing equivalence of APSD profiles of T and R OIDPs. We propose here a modified version of the CSRS (mCSRS) and evaluated systematically its behavior when T and R CI profiles were identical. Different scenarios comprising CI profiles with different number of deposition sites and shapes were generated by Monte-Carlo simulation. For each scenario, the mCSRS was applied to 20,000 independent sets of 30 T and 30 R CI profiles that were identical. Different metrics (including mean and median) of the distribution of 900 mCSRSs (30 T?×?30 R) were then evaluated for their suitability as a test statistic (i.e., independent of the number of sites and shape of the CI profile) for APSD equivalence testing. The median of the distribution of 900 mCSRSs (MmCSRS) was one regardless of the number of sites and shape of the CI profile. Hence, the MmCSRS is a robust metric for CI profile equivalence testing when T and R CI profiles are identical and potentially useful for APSD equivalence testing.     

Reconstitution and functional analyses of neutrophils and distinct subsets of monocytes after allogeneic stem cell transplantation

Purpose

The aim of the study was to investigate the recovery of the innate immune system within the first 100?days after allogeneic peripheral blood stem cell transplantation (PBSCT) and to elucidate a potential correlation with such important events as severe infectious complications or graft-versus-host disease (GvHD).

Methods

In 30 consecutive patients who underwent allogeneic PBSCT, absolute numbers of neutrophils and monocytes were determined and different functional analyses performed at different time points (day +30, +60 and +90, respectively). The capacity to phagocyte Escherichia coli (E. coli) as well as the induction of oxidative burst after incubation with different stimuli (Phorbol-12-myristate-13-acetate; PMA, the chemotactic peptide N-formyl-Met-Leu-Phe; f-MLP or opsonized E. coli) were analysed after engraftment.

Results

There was a rapid reconstitution concerning the capability of both neutrophils and monocytes to phagocyte E. coli without a significant increase between day +30 and +90. In contrast, a twofold increase of monocyte oxidative burst after incubation with PMA at day +90 was observed (P?=?0.017). Furthermore, the ability of neutrophils to induce oxidative burst after ingestion with E. coli was impaired on day +30 with a significant functional reconstitution on day +60 (P?=?0.01). The oxidative burst activity following incubation with f-MLP did not show significant changes after stem cell engraftment. Analysis of numeric reconstitution of CD14+CD16+ monocytes demonstrated a potential correlation with a decreased incidence of chronic GvHD.

Conclusion

The functional recovery of neutrophils and monocytes in the early period after allogeneic PBSCT differs not only concerning phagocytosis and oxidative burst but also with respect to the stimulus and the cell population that was analysed for oxidative burst activity. The subset of CD16+CD14+ monocytes might be a predictor for a reduced risk of chronic GvHD.