Proteomic Profiling of Serological Responses to Aspergillus fumigatus Antigens in Patients with Invasive Aspergillosis.

Aspergillus fumigatus is the species that most commonly causes the opportunistic infection invasive aspergillosis (IA) in patients being treated for hematological malignancies. Little is known about the A. fumigatus proteins that trigger the production of Aspergillus-specific IgG antibodies during the course of IA. To characterize the serological response to A. fumigatus protein antigens, mycelial proteins were separated by 2-D gel electrophoresis. The gels were immunoblotted with sera from patients with probable and proven IA and control patients without IA. We identified 49 different fungal proteins, which gave a positive IgG antibody signal. Most of these antigens play a role in primary metabolism and stress responses. Overall, our analysis identified 18 novel protein antigens from A. fumigatus. To determine whether these antigens can be used as diagnostic or prognostic markers or exhibit a protective activity, we employed supervised machine learning with decision trees. We identified two candidates for further analysis, the protein antigens CpcB and Shm2. Heterologously produced Shm2 induced a strongly proinflammatory response in human peripheral blood mononuclear cells after in vitro stimulation. In contrast, CpcB did not activate the immune response of PBMCs. These findings could serve as the basis for the development of an immunotherapy of IA.

Combined real-time PCR and galactomannan surveillance improves diagnosis of invasive aspergillosis in high risk patients with haematological malignancies.

Invasive aspergillosis (IA) is a leading complication of intensive treatment for haematological malignancies. Earlier diagnosis should facilitate effective antifungal therapy and prevent progression to invasive disease, which is often lethal. Polymerase chain reaction (PCR) assays, targeting the 28S and ITS ribosomal gene regions respectively, were evaluated for early detection of Aspergillus DNA and for diagnostic utility in patients receiving treatment in two busy haematopoietic stem cell transplant centres. Patients undergoing intensive chemotherapy, autologous or allogeneic transplant were eligible for inclusion in the study. EDTA blood and serum samples for circulating Aspergillus biomarkers, including galactomannan (GM), were collected twice-weekly on a prospective basis from all study patients who were categorized according to international consensus criteria for defining invasive fungal disease (IFD). Of 278 patients recruited there were 44 probable IA cases and only one proven case. Moderate sensitivity and specificity, poor positive predictive value (50-80%), but good negative predictive value (>80-90%) were common to both PCR assays. Overall biomarker performance could be improved by combining positive results of either PCR assay with GM taken within a 12-d period. The addition of PCR to GM monitoring in high-risk patients with haematological malignancies provides greater diagnostic accuracy in invasive aspergillosis.

Molecular diagnostic criteria of myeloproliferative neoplasms.

INTRODUCTION: Myeloproliferative neoplasms (MPN) are a heterogeneous group of clonal hematopoietic stem cell neoplasms characterized by the driver mutations JAK2, CALR, and MPL. These mutations cause constitutive activation of JAK-STAT signaling, which is central to pathogenesis of MPNs. Next-generation sequencing has further expanded the molecular landscape allowing for improved diagnostics, prognostication, and targeted therapy. AREAS COVERED: This review aims to address current understanding of the molecular diagnosis of MPN not only through improved awareness of the driver mutations but also the disease modifying mutations. In addition, other genetic factors such as clonal hematopoiesis of indeterminate potential (CHIP), order of mutation, and mutation co-occurrence are discussed and how these factors influence disease initiation and ultimately progression. How this molecular information is incorporated into risk stratification models allowing for earlier intervention and targeted therapy in the future will be addressed further. EXPERT OPINION: The genomic landscape of the MPN has evolved in the last 15 years with integration of next-generation sequencing becoming the gold standard of MPN management. Although diagnostics and prognostication have become more personalized, additional studies are required to translate these molecular findings into targeted therapy therefore improving patient outcomes.

FLT3 inhibitors as MRD-guided salvage treatment for molecular failure in FLT3 mutated AML.

Patients with FLT3-mutated AML have a high relapse rate and suboptimal outcomes. Many have co-mutations suitable for measurable residual disease (MRD) monitoring by RT-qPCR and those destined to relapse can be identified by high or rising levels of MRD, called molecular failure.  This provides a window for pre-emptive intervention, but there is little evidence to guide treatment. The use of FLT3 inhibitors (FLT3i) appears attractive but their use has not yet been evaluated.  We identified 56 patients treated with FLT3i at molecular failure.  The FLT3 mutation was an ITD in 52, TKD in 7 and both in 3. Over half of patients had previously received midostaurin. Molecular failure occurred at a median 9.2 months from diagnosis and was treated with gilteritinib (n = 38), quizartinib (n = 7) or sorafenib (n = 11). 60% achieved a molecular response, with 45% reaching MRD negativity. Haematological toxicity was low, and 22 patients were bridged directly to allogeneic transplant with another 6 to donor lymphocyte infusion. 2-year overall survival was 80% (95%CI 69-93) and molecular event-free survival 56% (95%CI 44-72). High-sensitivity next-generation sequencing for FLT3-ITD at molecular failure identified patients more likely to benefit. FLT3i monotherapy for molecular failure is a promising strategy which merits evaluation in prospective studies.

Allogeneic haematopoietic cell transplantation for myelofibrosis: proposed definitions and management strategies for graft failure, poor graft function and relapse: best practice recommendations of the EBMT Chronic Malignancies Working Party.

Allogeneic haematopoietic cell transplantation (allo-HCT) remains the only curative approach in myelofibrosis (MF). Despite advances over recent decades, relapse and non-relapse mortality rates remain significant. Relapse rates vary between 15 and 25% across retrospective studies and management strategies vary widely, ranging from palliation to adoptive immunotherapy and, in some cases, a second allo-HCT. Moreover, in allo-HCT, there is a higher incidence of poor graft function and graft failure due to splenomegaly and a hostile "pro-inflammatory" marrow niche. The Practice Harmonisation and Guidelines subcommittee of the Chronic Malignancies Working Party (CMWP) of EBMT convened an international panel consisting of transplant haematologists, histopathologists and molecular biologists to propose practical, clinically relevant definitions of graft failure, poor graft function and relapse as well as management strategies following allo-HCT. A systematic approach to molecular monitoring, histopathological assessment and chimerism testing is proposed. These proposed recommendations aim to increase the accuracy and uniformity of reporting and to thereby facilitate the development of more consistent approaches to these challenging issues. In addition, we propose management strategies for these complications.

Treatment-free remission following frontline nilotinib in patients with chronic phase chronic myeloid leukemia: 5-year update of the ENESTfreedom trial.

The ENESTfreedom trial assessed the feasibility of treatment-free remission (TFR) in patients with chronic myeloid leukemia in chronic phase (CML-CP) following frontline nilotinib treatment. Results for long-term outcomes after a 5-year follow-up are presented herein. Patients who had received ≥2 years of frontline nilotinib therapy and achieved MR4.5 underwent a 1-year nilotinib treatment consolidation phase before attempting TFR. At the 5-year data cut-off, 81/190 patients entering the TFR phase (42.6%) were still in TFR, with 76 (40.0%) in MR4.5. Patients who lost major molecular response (MMR) entered a treatment re-initiation phase; 90/91 patients entering this phase (98.9%) regained MMR and 84/91 patients (92.3%) regained MR4.5. The Kaplan-Meier estimated treatment-free survival rate at 5 years was 48.2%. No disease progression or CML-related deaths were reported. Whereas the incidence of adverse events (AEs) declined from 96 weeks following the start of TFR, an increase in AE frequency was observed for patients in the treatment re-initiation phase. Low Sokal risk score, BCR-ABL1IS levels at 48 weeks of TFR and stable MR4.5 response for the first year of TFR were associated with higher TFR rates. Overall, these results support the efficacy and safety of attempting TFR following upfront nilotinib therapy of >3 years in patients with CML-CP.

Impact of prior JAK-inhibitor therapy with ruxolitinib on outcome after allogeneic hematopoietic stem cell transplantation for myelofibrosis: a study of the CMWP of EBMT.

JAK1/2 inhibitor ruxolitinib (RUX) is approved in patients with myelofibrosis but the impact of pretreatment with RUX on outcome after allogeneic hematopoietic stem cell transplantation (HSCT) remains to be determined. We evaluated the impact of RUX on outcome in 551 myelofibrosis patients who received HSCT without (n = 274) or with (n = 277) RUX pretreatment. The overall leukocyte engraftment on day 45 was 92% and significantly higher in RUX responsive patients than those who had no or lost response to RUX (94% vs. 85%, p = 0.05). The 1-year non-relapse mortality was 22% without significant difference between the arms. In a multivariate analysis (MVA) RUX pretreated patients with ongoing spleen response at transplant had a significantly lower risk of relapse (8.1% vs. 19.1%; p = 0.04)] and better 2-year event-free survival (68.9% vs. 53.7%; p = 0.02) in comparison to patients without RUX pretreatment. For overall survival the only significant factors were age > 58 years (p = 0.03) and HLA mismatch donor (p = 0.001). RUX prior to HSCT did not negatively impact outcome after transplantation and patients with ongoing spleen response at time of transplantation had best outcome.

The vascular targeting agent combretastatin-A4 and a novel cis-Restricted {beta}-Lactam Analogue, CA-432, induce apoptosis in human chronic myeloid leukemia cells and ex vivo patient samples including those displaying multidrug resistance.

Combretastatin-A4 (CA-4) is a natural derivative of the African willow tree Combretum caffrum. CA-4 is one of the most potent antimitotic components of natural origin, but it is, however, intrinsically unstable. A novel series of CA-4 analogs incorporating a 3,4-diaryl-2-azetidinone (ß-lactam) ring were designed and synthesized with the objective to prevent cis -trans isomerization and improve the intrinsic stability without altering the biological activity of CA-4. Evaluation of selected ß-lactam CA-4 analogs demonstrated potent antitubulin, antiproliferative, and antimitotic effects in human leukemia cells. A lead ß-lactam analog, CA-432, displayed comparable antiproliferative activities with CA-4. CA-432 induced rapid apoptosis in HL-60 acute myeloid leukemia cells, which was accompanied by depolymerization of the microtubular network, poly(ADP-ribose) polymerase cleavage, caspase-3 activation, and Bcl-2 cleavage. A prolonged G(2)M cell cycle arrest accompanied by a sustained phosphorylation of mitotic spindle checkpoint protein, BubR1, and the antiapoptotic proteins Bcl-2 and Bcl-x(L) preceded apoptotic events in K562 chronic myeloid leukemia (CML) cells. Molecular docking studies in conjunction with comprehensive cell line data rule out CA-4 and ß-lactam derivatives as P-glycoprotein substrates. Furthermore, both CA-4 and CA-432 induced significantly more apoptosis compared with imatinib mesylate in ex vivo samples from patients with CML, including those positive for the T315I mutation displaying resistance to imatinib mesylate and dasatinib. In summary, synthetic intrinsically stable analogs of CA-4 that display significant clinical potential as antileukemic agents have been designed and synthesized.

Myelodysplastic Syndrome/Acute Myeloid Leukemia Arising in Idiopathic Erythrocytosis.

The term "idiopathic erythrocytosis (IE)" is applied to those cases where a causal clinical or pathological event cannot be elucidated and likely reflects a spectrum of underlying medical and molecular abnormalities. The clinical course of a patient with IE is described manifesting as a persistent erythrocytosis with a low serum erythropoietin level, mild eosinophilia, and with evidence of a thrombotic event. The patient subsequently developed a myelodysplasic syndrome (MDS) and acute myeloid leukemia (AML), an event not observed in erythrocytosis patients other than those with polycythemia vera (PV). Application of a next-generation sequencing (NGS) approach targeted for myeloid malignancies confirmed wild-type JAK2 exons 12-15 and identified a common SH2B3 W262R single-nucleotide polymorphism associated with the development of hematological features of myeloproliferative neoplasms (MPNs). Further NGS analysis detected a CBL L380P mutated clone expanding in parallel with the development of MDS and subsequent AML. Despite the absence of JAK2, MPL exon 10, or CALR exon 9 mutations, a similarity with the disease course of PV/MPN was evident. A clonal link between the erythrocytosis and AML could be neither confirmed nor excluded. Future molecular identification of the mechanisms underlying IE is likely to provide a more refined therapeutic approach.

Development of a Targeted Next-Generation Sequencing Assay to Detect Diagnostically Relevant Mutations of JAK2, CALR, and MPL in Myeloproliferative Neoplasms.

BACKGROUND: The classical Philadelphia chromosome-negative myeloproliferative neoplasms (MPNs), consisting of polycythemia vera, essential thrombocythemia, and primary myelofibrosis, are a heterogeneous group of neoplasms that harbor driver mutations in the JAK2, CALR, and MPL genes. The detection of mutations in these genes has been incorporated into the recent World Health Organization (WHO) diagnostic criteria for MPN. Given a pressing clinical need to screen for mutations in these genes in a routine diagnostic setting, a targeted next-generation sequencing (NGS) assay for the detection of MPN-associated mutations located in JAK2 exon 14, JAK2 exon 12, CALR exon 9, and MPL exon 10 was developed to provide a single platform alternative to reflexive, stepwise diagnostic algorithms. METHODS: Polymerase chain reaction (PCR) primers were designed to target mutation hotspots in JAK2 exon 14, JAK2 exon 12, MPL exon 10, and CALR exon 9. Multiplexed PCR conditions were optimized by using qualitative PCR followed by NGS. Diagnostic genomic DNA from 35 MPN patients, known to harbor driver mutations in one of the target genes, was used to validate the assay. RESULTS: One hundred percent concordance was observed between the previously-identified mutations and those detected by NGS, with no false positives, nor any known mutations missed (specificity = 100%, CI = 0.96, sensitivity = 100%, CI = 0.89). Improved resolution of mutation sequences was also revealed by NGS analysis. CONCLUSION: Detection of diagnostically relevant driver mutations of MPN is enhanced by employing a targeted multiplex NGS approach. This assay presents a robust solution to classical MPN mutation screening, providing an alternative to time-consuming sequential analyses.