Identification of recurrent driver mutations in genes encoding tyrosine kinases has resulted in the development of molecularly targeted strategies designed to improve the outcomes for patients diagnosed with acute myeloid leukemia (AML). The receptor tyrosine kinase FLT3, is the most commonly mutated gene in AML, with internal tandem duplications within the juxtamembrane domain (FLT3-ITD) or missense mutations in the tyrosine kinase domain (FLT3-TKD), present in 30%-35% of AML patients at diagnosis. An established driver mutation and marker of poor prognosis, the FLT3 tyrosine kinase has emerged as an attractive therapeutic target, and thus has encouraged the development of FLT3 tyrosine kinase inhibitors (TKIs). However, the therapeutic benefit of FLT3 inhibition, particularly as monotherapy, frequently results in the development of treatment resistance and disease relapse. Commonly, FLT3 inhibitor resistance is induced by the emergence of secondary lesions in the FLT3 gene, particularly in the second tyrosine kinase domain at residue Asp835 (D835) to form a ‘dual mutation’ (ITD-D835). Individual FLT3-ITD and FLT3-TKD mutations influence independent signaling cascades however, currently little is known which divergent signaling pathways are controlled by each of these FLT3 specific mutations, particularly in the context of patients harboring dual ITD-D835 mutations. This review provides a comprehensive analysis of the known discrete and cooperative signaling pathways regulated by each of the FLT3 specific mutations, as well as the therapeutic approaches that hold the most promise for development of more durable and personalized therapeutic approaches targeting mutant FLT3, to improve the treatment of AML.
We would like to update readers on the treatment landscape for severe chronic immune thrombocytopenia (ITP) in the wake of recent welcome changes to the Pharmaceutical Benefits Scheme (PBS) reimbursement criteria in Australia and since the publication of our consensus treatment guidelines.1 First, splenectomy is no longer required to access thrombopoietin receptor agonists (TPO-RAs) such as romplostim and eltrombopag. This change aligns with management guidelines and clinical data showing no difference in outcome between patients with splenectomy versus those without splenectomy receiving TPO-RAs and permits ITP treatment selection best tailored towards patient preferences and clinical factors.1-4 Second, a new TPO-RA avatrombopag is now PBS listed as an alternative to romiplostim and eltrombopag. There are no head-to-head data to suggest superiority of one TPO-RA over another, but differing administration routes, dose schedules, interactions and side effect profiles may assist clinician–patient decision-making with treatment selection. Table 1 highlights the main differences between the available TPO-RA agents. Bone marrow reticulin Thrombosis Rebound thrombocytopenia Risk of progression of myeloid malignancies Chelates polyvalent cations (such as calcium and iron) OATP1B1 and BCRP substrates caution (e.g. ciclosporin) Reduce statin dose Hepatotoxicity Cataracts Thrombosis Rebound thrombocytopenia Bone marrow reticulin Risk of progression of myeloid malignancies CYP2C9 and CYP3A4 inhibitors may increase avatrombopag levels (e.g. fluconazole) CYP2C9 and CYP3A4 inducers may reduce avatrombopag levels (e.g. rifampicin) Bone marrow reticulin Thrombosis Rebound thrombocytopenia Risk of progression of myeloid malignancies Third, switching between TPO-RAs for any reason including tolerance or failure is now permissible at any time. Previously, switching was not allowed after 24 weeks of therapy, even after the development of an unanticipated adverse event or eventual loss of response. Although one may anticipate an equivalent TPO-RA class effect with these agents, retrospective data demonstrate switching between these drugs for inadequate responses can be successful with improved platelet counts and reductions in concomitant medications.5, 6 Thus, with the relaxation of switching rules and the PBS listing of avatrombopag, we now have an additional line of therapy rather than just another oral alternative to romiplostim or eltrombopag. Fourth, a clinical response as determined by the treating physician is sufficient to warrant ongoing PBS reimbursement in lieu of absolute platelet targets that were insensitive to clinically apparent improvements in bleeding and quality of life (QoL). Previously, the threshold for some patients to meet platelet response criteria devised for clinical trial environments was extremely difficult to achieve for PBS reimbursement purposes, even though there was clear benefit for patients in terms of reduced bleeding, improved platelet counts and lower concomitant ITP therapies. Along with penalising patients who needed intermittent rescue therapies, these measures had seemed to restrict TPO-RA access for the patients with the most severe disease. This has now been rectified as the threshold for ongoing use is clinically determined by the treating physician most cognisant of their patients' medical priorities. Finally, rituximab (intravenous B-cell depleting anti-CD20 monoclonal antibody) is now unrestricted on the PBS. Previously, off-label rituximab use was either self-funded or compassionately supported with a bias towards lower dosing schedules that were more cost-effective.7 Any new enthusiasm for PBS-reimbursed rituximab is probably balanced by recent coronavirus disease 2019 (COVID-19) pandemic concerns and tempered by previous analyses demonstrating only a modest long-term success rate, particularly when administered in monotherapy.8, 9 There are still many uncertainties ahead for optimal ITP management such as targeting the natural history of ITP before it becomes chronic, exploring the role of TPO-RAs in earlier stages of ITP, predicting and targeting patients at the highest risk of bleeding and rationally drafting the optimal sequence or combination of novel and existing therapies for severely refractory patients. Data from the iWISh survey suggest that patients prefer to halt the progression or worsening of their ITP above QoL and bleeding, in contradistinction to their physicians.10 The ultimate goal for most patients is to optimise initial therapy of newly diagnosed ITP to prevent progression to the chronic phase. The recent FLIGHT (Newly Diagnosed Immune Thrombocytopenia Testing the Standard Steroid Treatment Against Combined Steroid & Mycophenolate) study, which explored the addition of mycophenolate mofetil to corticosteroids in first-line therapy, demonstrated an improved response and a halving of the progression rate to chronic ITP. This was at the expense of worsened patient-reported QoL measures at the end of the study11; however, it seems reasonable to expect that the longer-term impact on cumulative disease and treatment burden by preventing the development of chronic ITP will eventually lead to better QoL. Treatment strategies addressing the pervasive but insidious symptom of fatigue in ITP are also lacking. Patients with newly diagnosed ITP remain vulnerable to the uncertainties of their diagnosis, anxious about their prognosis, and still have limited treatment options available to them. Despite these therapeutic challenges, we welcome these updates to the PBS listings for the TPO-RAs. We look forward to the promise of these and other novel agents in the modern treatment landscape ahead. Amongst newer therapies, fostamatinib (spleen associated tyrosine kinase inhibitor) was listed by the Food and Drug Administration for chronic ITP in 2018 but remains unavailable in Australia. Clinical trials in ITP have recently been completed for rilzabrutinib (Bruton's tyrosine kinase [BTK] inhibitor) and efgartigimod (neonatal Fc receptor [FcRn] inhibitor).12, 13 As new clinical trials are opening across Australia now with B-cell activating factor [BAFF] and a proliferation-inducing ligand [APRIL] inhibitors, the prospect of better targeting the immune lesion in ITP beckons.
Microparticles (MP) are small fragments of cytoplasm shed from a cell surface and their role in the pathophysiology of disease is being extensively investigated. A novel staining technique for quantifying total MP in peripheral blood was evaluated in this study. Evaluation of Bodipy-maleimide (or bio-maleimide) as a stain for quantifying total MP in peripheral blood by flow cytometry. Samples were obtained from 10 healthy donors after informed consent. Plasma was prepared by sequential centrifugation at 1500 g followed by 13,000 g and stained with Annexin V and bio-maleimide. Enumeration beads were added after 15 min of incubation with the stain and samples analyzed on a FACS Canto flow cytometer. Detection and quantification of MP by bio-maleimide staining was comparable with that by Annexin V. The total mean MP level with bio-maleimide staining was 34 +/- 19.7/microl (range of 11.6-68.1/microl) and with Annexin V staining it was 38.9 +/- 29.8/microl (range of 10.6 to 112.9/microl). There was no significant difference using a paired t-test and methods were comparable using a Bland-Altman plot. Bio-maleimide is a useful and inexpensive stain to measure total MP levels in peripheral blood by flow cytometry. This technique could be employed to study thrombotic risks in a variety of disease states.
Enumeration of circulating microvesicles (MVs) by conventional flow cytometry is accomplished by the addition of a known amount of counting beads and calculated from the formula: MV/μl = (MV count/bead count) × final bead concentration. We sought to optimize each variable in the equation by determining the best parameters for detecting 'MV count' and examining the effects of different bead preparations and concentrations on the final calculation. Three commercially available bead preparations (TruCount, Flow-Count and CountBright) were tested, and MV detection on a BD FACSCanto was optimized for gating by either forward scatter (FSC) or side scatter (SSC); the results were compared by calculating different subsets of MV on a series of 74 typical patient plasma samples. The relationship between the number of beads added to each test and the number of beads counted by flow cytometry remained linear over a wide range of bead concentrations (R2 ≥ 0.997). However, TruCount beads produced the most consistent (concentration variation = 3.8%) calculated numbers of plasma CD41+/Annexin V+ MV, which were significantly higher from that calculated using either Flow-Count or CountBright (p < 0.001). The FACSCanto was able to resolve 0.5 μm beads by FSC and 0.16 μm beads by SSC, but there were significantly more background events using SSC compared with FSC (3113 vs. 470; p = 0.008). In general, sample analysis by SSC resulted in significantly higher numbers of MV (p < 0.0001) but was well correlated with enumeration by FSC for all MV subtypes (ρ = 0.62-0.89, p < 0.0001). We conclude that all counting beads provided linear results at concentrations ranging from 6 beads/μl to 100 beads/μl, but TruCount was the most consistent. Using SSC to gate MV events produced high background which negatively affected counting bead enumeration and overall MV calculations. Strategies to reduce SSC background should be employed in order to reliably use this technique.
The identification of recurrent driver mutations in genes encoding tyrosine kinases has resulted in the development of molecularly-targeted treatment strategies designed to improve outcomes for patients diagnosed with acute myeloid leukemia (AML). The receptor tyrosine kinase FLT3 is the most commonly mutated gene in AML, with internal tandem duplications within the juxtamembrane domain (FLT3-ITD) or missense mutations in the tyrosine kinase domain (FLT3-TKD) present in 30–35% of AML patients at diagnosis. An established driver mutation and marker of poor prognosis, the FLT3 tyrosine kinase has emerged as an attractive therapeutic target, and thus, encouraged the development of FLT3 tyrosine kinase inhibitors (TKIs). However, the therapeutic benefit of FLT3 inhibition, particularly as a monotherapy, frequently results in the development of treatment resistance and disease relapse. Commonly, FLT3 inhibitor resistance occurs by the emergence of secondary lesions in the FLT3 gene, particularly in the second tyrosine kinase domain (TKD) at residue Asp835 (D835) to form a ‘dual mutation’ (ITD-D835). Individual FLT3-ITD and FLT3-TKD mutations influence independent signaling cascades; however, little is known about which divergent signaling pathways are controlled by each of the FLT3 specific mutations, particularly in the context of patients harboring dual ITD-D835 mutations. This review provides a comprehensive analysis of the known discrete and cooperative signaling pathways deregulated by each of the FLT3 specific mutations, as well as the therapeutic approaches that hold the most promise of more durable and personalized therapeutic approaches to improve treatments of FLT3 mutant AML.
Blastic plasmacytoid dendritic cell neoplasm (BPDCN) is a rare and aggressive haematological malignancy in the elderly, with a high frequency of cutaneous and bone marrow involvement and poor prognosis. We report a case of BPDCN with classic presentation and discuss its treatment and the value of different investigation tools used in diagnosis and response assessment.
Abstract Introduction Variants of uncertain significance (VUS) are commonly reported in cancer with the widespread adoption of diagnostic massive parallel sequencing. The rate of reclassification of VUS in patients with haematological malignancy is not known and we evaluated this retrospectively. We also investigated whether re‐evaluating VUS in 12–24 months or greater than 24 months post‐initial classification was significant. Method A retrospective audit of patients with haematological malignancies referred to the Molecular Medicine Department at the John Hunter Hospital in Newcastle, Australia between September 2018 and December 2021. Data was analysed for VUS, which was then re‐analysed in standard software using current somatic variant guidelines. Proportions of VUS at baseline were compared to post‐re‐analysis. Results The most common diagnoses in the patient cohort ( n = 944) were acute myelogenous leukaemia (41%), myelodysplastic syndrome (31%), and chronic myelomonocytic leukaemia (7%). A total of 210 VUS were re‐analysed. The most common VUS were in the TET2 (20%), RUNX1 (10%) and DNMT3A (9%) genes. A total of 103 were re‐analysed at 24–39 months post‐initial classification and 107 variants were re‐analysed between 12 and 24 months post‐initial classification. Of these, 33 (16%) of VUS were re‐classified at 24–39 months and 12 (11%) were re‐classified at 12–24 months post‐initial classification. The most common variants that were re‐classified in both groups were CSF3R (32%), TET2 (29%), ASXL1 (11%) and ZRSR2 (11%). Conclusion This study on reclassification of VUS in blood cancers demonstrated that one in seven VUS were re‐classified 12 months post initial classification. This can inform practice guidelines and potentially impact the prognosis, diagnosis and treatment of haematological malignancies.
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