ROP-ET: a prospective phase III trial investigating the efficacy and safety of ropeginterferon alfa-2b in essential thrombocythemia patients with limited treatment options.

Interferon-based therapies, such as ropeginterferon alfa-2b have emerged as promising disease-modifying agents for myeloproliferative neoplasms (MPNs), including essential thrombocythemia (ET). Current ET treatments aim to normalize hematological parameters and reduce the thrombotic risk, but they do not modify the natural history of the disease and hence, have no impact on disease progression. Ropeginterferon alfa-2b (trade name BESREMi®), a novel, monopegylated interferon alfa-2b with an extended administration interval, has demonstrated a robust and sustained efficacy in polycythemia vera (PV) patients. Given the similarities in disease pathophysiology and treatment goals, ropeginterferon alfa-2b holds promise as a treatment option for ET. The ROP-ET trial is a prospective, multicenter, single-arm phase III study that includes patients with ET who are intolerant or resistant to, and/or are ineligible for current therapies, such as hydroxyurea (HU), anagrelide (ANA), busulfan (BUS) and pipobroman, leaving these patients with limited treatment options. The primary endpoint is a composite response of hematologic parameters and disease-related symptoms, according to modified European LeukemiaNet (ELN) criteria. Secondary endpoints include improvements in symptoms and quality of life, molecular response and the safety profile of ropeginterferon alfa-2b. Over a 3-year period the trial assesses longer term outcomes, particularly the effects on allele burden and clinical outcomes, such as disease-related symptoms, vascular events and disease progression. No prospective clinical trial data exist for ropeginterferon alfa-2b in the planned ET study population and this study will provide new findings that may contribute to advancing the treatment landscape for ET patients with limited alternatives. TRIAL REGISTRATION: EU Clinical Trials Register; EudraCT, 2023-505160-12-00; Registered on October 30, 2023.

Comparison of acute myeloid leukemia and myelodysplastic syndromes with TP53 aberrations.

TP53 aberrations are found in approximately 10% of patients with acute myeloid leukemia (AML) and myelodysplastic syndromes (MDS) and are considered early driver events affecting leukemia stem cells. In this study, we compared features of a total of 84 patients with these disorders seen at a tertiary cancer center. Clinical and cytogenetic characteristics as well as immunophenotypes of immature blast cells were similar between AML and MDS patients. Median overall survival (OS) was 226 days (95% confidence interval [CI], 131-300) for the entire cohort with an estimated 3-year OS rate of 11% (95% CI, 6-22). OS showed a significant difference between MDS (median, 345 days; 95% CI, 235-590) and AML patients (median, 91 days; 95% CI, 64-226) which is likely due to a different co-mutational pattern as revealed by next-generation sequencing. Transformation of TP53 aberrant MDS occurred in 60.5% of cases and substantially reduced their survival probability. Cox regression analysis revealed treatment class and TP53 variant allele frequency as prognostically relevant parameters but not the TP53-specific prognostic scores EAp53 and RFS. These data emphasize similarities between TP53 aberrant AML and MDS and support previous notions that they should be classified and treated as a distinct disorder.

Incidence of invasive fungal infections in patients with hematological malignancies receiving ibrutinib therapy in south-east Austria.

Since the broad implementation of ibrutinib therapy, an increasing number of studies have been reported on invasive fungal infections (IFI) associated with ibrutinib administration. We conducted a retrospective cohort study in three hospitals in south-east Austria in order to assess the local epidemiology of ibrutinib associated IFIs. A total of 113 patients with underlying hematological malignancy were included in the study. During the study period, a single IFI episode was observed, which corresponds to an IFI incidence of 2.3 cases per 100 person years (95% CI: 0.12-11.47). IFIs during ibrutinib therapy seem to be a rare event in case of absent additional risk factors for IFIs.

Ibrutinib is an effective drug used to treat a variety of blood cancers, but it might increase risk for life-threatening invasive fungal infections (IFIs). In our study, a low number (1 IFI per 43 patient years) of patients on ibrutinib developed an IFI.

Loss of RAF kinase inhibitor protein is involved in myelomonocytic differentiation and aggravates RAS-driven myeloid leukemogenesis.

RAS-signaling mutations induce the myelomonocytic differentiation and proliferation of hematopoietic stem and progenitor cells. Moreover, they are important players in the development of myeloid neoplasias. RAF kinase inhibitor protein (RKIP) is a negative regulator of RAS-signaling. As RKIP loss has recently been described in RAS-mutated myelomonocytic acute myeloid leukemia, we now aimed to analyze its role in myelomonocytic differentiation and RAS-driven leukemogenesis. Therefore, we initially analyzed RKIP expression during human and murine hematopoietic differentiation and observed that it is high in hematopoietic stem and progenitor cells and lymphoid cells but decreases in cells belonging to the myeloid lineage. By employing short hairpin RNA knockdown experiments in CD34+ umbilical cord blood cells and the undifferentiated acute myeloid leukemia cell line HL-60, we show that RKIP loss is indeed functionally involved in myelomonocytic lineage commitment and drives the myelomonocytic differentiation of hematopoietic stem and progenitor cells. These results could be confirmed in vivo, where Rkip deletion induced a myelomonocytic differentiation bias in mice by amplifying the effects of granulocyte macrophage-colony-stimulating factor. We further show that RKIP is of relevance for RAS-driven myelomonocytic leukemogenesis by demonstrating that Rkip deletion aggravates the development of a myeloproliferative disease in NrasG12D -mutated mice. Mechanistically, we demonstrate that RKIP loss increases the activity of the RAS-MAPK/ERK signaling module. Finally, we prove the clinical relevance of these findings by showing that RKIP loss is a frequent event in chronic myelomonocytic leukemia, and that it co-occurs with RAS-signaling mutations. Taken together, these data establish RKIP as novel player in RAS-driven myeloid leukemogenesis.

Sensitive and broadly applicable residual disease detection in acute myeloid leukemia using flow cytometry-based leukemic cell enrichment followed by mutational profiling.

Persistent measurable residual disease (MRD) is an increasingly important prognostic marker in acute myeloid leukemia (AML). Currently, MRD is determined by multi-parameter flow cytometry (MFC) or PCR-based methods detecting leukemia-specific fusion transcripts and mutations. However, while MFC is highly operator-dependent and difficult to standardize, PCR-based methods are only available for a minority of AML patients. Here we describe a novel, highly sensitive and broadly applicable method for MRD detection by combining MFC-based leukemic cell enrichment using an optimized combinatorial antibody panel targeting CLL-1, TIM-3, CD123 and CD117, followed by mutational analysis of recurrently mutated genes in AML. In dilution experiments this method showed a sensitivity of 10-4 to 10-5 for residual disease detection. In prospectively collected remission samples this marker combination allowed for a median 67-fold cell enrichment with sufficient DNA quality for mutational analysis using next generation sequencing (NGS) or digital PCR in 39 out of 41 patients. Twenty-one samples (53.8%) tested MRD positive, whereas 18 (46.2%) were negative. With a median follow-up of 559 days, 71.4% of MRD positive (15/21) and 27.8% (5/18) of MRD negative patients relapsed (P = .007). The cumulative incidence of relapse (CIR) was higher for MRD positive patients (5-year CIR: 90.5% vs 28%, P < .001). In multivariate analysis, MRD positivity was a prominent factor for CIR. Thus, MFC-based leukemic cell enrichment using antibodies against CLL-1, TIM-3, CD123 and CD117 followed by mutational analysis allows high sensitive MRD detection and is informative on relapse risk in the majority of AML patients.

Clinical implications of subclonal TP53 mutations in acute myeloid leukemia.

The role of subclonal TP53 mutations, defined by a variant allele frequency of <20%, has not been addressed in acute myeloid leukemia yet. We, therefore, analyzed their prognostic value in a cohort of 1,537 patients with newly diagnosed disease, prospectively treated within three trials of the "German-Austrian Acute Myeloid Leukemia Study Group". Mutational analysis was performed by targeted deep sequencing and patients with TP53 mutations were categorized by their variant allele frequency into groups with frequencies >40%, 20%-40% and <20%. A total of 108 TP53 mutations were found in 98 patients (6.4%). Among these, 61 patients had variant allele frequencies >40%, 19 had variant allele frequencies between 20%-40% and 18 had frequencies <20%. Compared to specimens with clonal TP53 mutations, those with subclonal ones showed significantly fewer complex karyotypes and chromosomal losses. In either TP53-mutated group, patients experienced significantly fewer complete responses (P<0.001) and had worse overall and event-free survival rates (P<0.0001). In Cox regression analyses adjusting for age, white blood cell count, cytogenetic risk and type of acute myeloid leukemia, the adverse prognostic effect of TP53 mutations remained significant for all TP53-mutated subgroups. These data suggest that subclonal TP53 mutations are a novel prognostic parameter in acute myeloid leukemia and emphasize the usefulness of next-generation sequencing technologies for risk stratification in this disorder. The study was registered at ClinicalTrials.gov with number NCT00146120.

Antifungal Prophylaxis with Posaconazole Delayed-Release Tablet and Oral Suspension in a Real-Life Setting: Plasma Levels, Efficacy, and Tolerability.

We continuously determined posaconazole plasma concentrations (PPCs) in 61 patients with hematological malignancies receiving posaconazole (PCZ) delayed-release tablets (DRT; 48 patients; median duration of intake, 92 days) and PCZ oral solution (OS; 13 patients; median duration of intake, 124 days). PCZ DRT and OS antifungal prophylaxis was efficient and well tolerated. Thirty-four of 48 patients (71%) receiving DRT always had PPCs of >0.7 mg/liter, while 14 of 48 patients (29%) had at least one PPC of ≤0.7 mg/liter. In patients receiving OS, 4 of 13 patients (31%) always had PPCs of >0.7 mg/liter, 6 of 13 patients (46%) had at least one PPC of ≤0.7 mg/liter, and 3 (23%) patients never reached a PPC of 0.7 mg/liter. In patients with at least one determined PPC, the mean proportion of all PPCs of >0.7 mg/liter was 91% for PCZ DRT, whereas it was 52% for PCZ OS (P = 0.001). In the per sample analysis, PPCs were significantly more likely to be >0.7 mg/liter in patients receiving DRT than in patients receiving OS (PPCs were >0.7 mg/liter in 91.4% [297/325] of patients receiving DRT versus 70.3% [85/121] of patients receiving OS; P < 0.001). Patients receiving PCZ DRT had higher proportions of PPCs of >0.7 mg/liter than patients receiving OS both in the per patient and in the per sample analyses. Two patients (3%) had side effects during PCZ prophylaxis, and one (2%) had fungal breakthrough infection. Therapeutic drug monitoring enables detection of extended periods of PPCs of ≤0.7 mg/liter (e.g., due to nonadherence or graft-versus-host disease), which may also be associated with the loss of protective intracellular PCZ concentrations, regardless of the PCZ formulation.

Residual disease detection using targeted parallel sequencing predicts relapse in cytogenetically normal acute myeloid leukemia.

Despite achieving complete remission after intensive therapy, most patients with cytogenetically normal (CN) AML relapse due to the persistence of submicroscopic residual disease. In this pilot study, we hypothesized that detection of leukemia-specific mutations following consolidation treatment using a targeted parallel sequencing approach predicts relapse. We included 34 AML patients of whom diagnostic material and remission bone marrow slides after at least one cycle of consolidation were available. Isolated DNA was screened for mutations in 19 genes using an Ion Torrent sequencing platform. Furthermore, the variant allelic frequency of distinct mutations was validated by digital PCR and sequencing using a barcoding approach. Twenty-seven out of 34 patients could be analyzed for mutation clearance. We identified 68 somatic mutations at diagnosis (median, 3 mutations per patient; range 1-5) and 22 of these were still detected in 16 patients after consolidation therapy with a reliable sensitivity of 0.5% (median, 1 mutation; range 0-3). The most frequent noncleared mutations were found in DNMT3A. However, as persistence of these mutations has recently been shown to be without any impact on relapse risk, we performed survival and relapse risk analysis excluding DNMT3A mutations. Importantly, persistence of non-DNMT3A mutations was associated with a higher risk of AML relapse (7/8 pts versus 6/19 pts; P = .013) and with a shorter relapse-free survival (333 days vs. not reached; log-rank P = .0219). Detection of residual disease by routine targeted parallel sequencing proved feasible and effective as persistence of somatic mutations other than DNMT3A were prognostic for relapse in CN AML.

The frequency of occurrence of fish-shaped red blood cells in different haematologic disorders.

BACKGROUND: Red blood cells (RBC) resembling the silhouette of a fish are rarely observed in peripheral blood (PB) smears. In this study, we determined the frequency of occurrence of fish-shaped RBC in different haematologic diseases. METHODS: We examined PB smears of patients with iron deficiency anaemia (IDA) (n=23), ß-thalassaemia minor (BTM) (n=30), sickle cell disease (SCD) (n=7), autoimmune haemolytic anaemia (AIHA) (n=13), microangiopathic haemolytic anaemia (MAHA) (n=11), hereditary sphaerocytosis (HS) (n=4), hereditary elliptocytosis (HE) (n=3), vitamin B12 and folate deficiency (n=15), anaemia in liver disease (LD) (n=17), myelodysplastic syndrome (MDS) (n=15), acute myeloid leukaemia (AML) (n=29), chronic myeloid leukaemia (CML) (n=18), primary myelofibrosis (PMF) (n=12), chronic myelo-monocytic leukaemia (CMML) (n=15) and 21 healthy controls by light microscopy for the occurrence of fish-shaped erythrocytes. The fish-shaped RBC were counted as cells per 20 high-power fields (HPF) at 1000-fold magnification, and slides containing ≥1 fish-shaped RBC/20 HPF were regarded as positive. RESULTS: Fish-shaped RBC were significantly found in HE, iron deficiency, vitamin B12/folate deficiency, LD and PMF. The highest numbers of fish-shaped RBC were seen in HE and vitamin B12/folate deficiency. In patients with BTM, MDS, AML and CMML, this RBC anomaly was only occasionally observed. Furthermore, a statistically significant negative correlation of haemoglobin with the occurrence of fish-shaped RBC was apparent (p<0.014). CONCLUSIONS: Our data show that the occurrence of fish-shaped RBC is suggestive of a pathologic condition, especially IDA, HE, vitamin B12 or folate deficiency, primary mylofibrosis or LD, and is significantly associated with severity of anaemia.

Real-world challenges and unmet needs in the diagnosis and treatment of suspected invasive pulmonary aspergillosis in patients with haematological diseases: An illustrative case study.

Recent years have seen important advances in the diagnosis of invasive pulmonary aspergillosis (IPA), complemented by the introduction of new therapies. Despite this, IPA remains a major cause of infection-related mortality in patients with haematological diseases. There are two main reasons for this. First, diagnosis of IPA remains a challenge, since risk factors and the clinical, radiological and mycological presentations vary not only by fungal disease stage, but also by patient group (eg neutropenic vs non-neutropenic patients). Diagnosis is particularly challenging in patients receiving mould-active prophylactic or empirical treatment, which reduces the sensitivity of all diagnostic tests for IPA. Second, treatment of IPA is complex due to unpredictable pharmacokinetic profiles of antifungal agents, small therapeutic window in terms of exposure and adverse events, and multiple drug-drug interactions through the CYP450 system. Here we report a case of a 23-year-old male with severe aplastic anaemia and subpleural nodules. Diagnostic tests for IPA obtained during ongoing mould-active empirical treatment were negative. Intravenous voriconazole was stopped after visual disturbances and hallucinations. The patient then had an anaphylactic reaction to liposomal amphotericin B and was switched to intravenous posaconazole, which had to be discontinued due to a significant increase in transaminase levels. He was treated with oral isavuconazole with reduced dosage, triggered by increasing transaminases under the standard dosage. Even under reduced dosage, blood concentrations of isavuconazole were high and treatment was successful. The case illustrates real-world challenges and unmet needs in the diagnosis and treatment of IPA in patients with haematological diseases.

Early Hyperglycemia after Initiation of Glucocorticoid Therapy Predicts Adverse Outcome in Patients with Acute Graft-versus-Host Disease.

Because of first-line treatment with high-dose glucocorticoids (GC), steroid-induced hyperglycemia develops frequently in patients with acute graft-versus-host disease (aGVHD), potentially affecting their outcome. We performed a retrospective analysis on 104 patients who received systemic GC for aGVHD and investigated the consequences of aberrant glucose metabolism. In particular, we focused on glucose parameters early after initiation of GC. With a median of 50 (range, 4 to 513) blood glucose measurements during GC treatment, increasing mean, median, and maximum glucose levels and the need for insulin treatment were associated with decreased overall survival (OS) in simple and multiple survival analysis. Early hyperglycemia, as defined by mean blood glucose levels >125 mg/dL during the first 3 days of GC therapy, was also found to be highly associated with adverse outcome (hazard ratio [HR], 2.5 for death; 95% confidence interval [CI], 1.3 to 4.8, and HR of 3.5 for death due to nonrelapse mortality, 95% CI, 1.7 to 7.5, in a competing risk analysis). A score based on early hyperglycemia and nonresponse to GC within 7 days allowed the identification of 3 risk groups: patients with both risk factors had an inferior OS at 5 years of 4.1% compared with 75.4% in patients with none. Patients with 1 risk factor had a 5-year OS rate of 32.0% (P = .0002 for trend). Early hyperglycemia after GC initiation is a prominent risk factor for adverse outcome in patients with aGVHD. A score based solely on early hyperglycemia and lack of response to GC can predict survival in these patients.

Pre-fibrotic/early primary myelofibrosis vs. WHO-defined essential thrombocythemia: The impact of minor clinical diagnostic criteria on the outcome of the disease.

The 2016 revised WHO criteria for the diagnosis of pre-fibrotic/early primary myelofibrosis (pre-PMF) require at least one of the following four borderline expressed minor clinical criteria: anemia, leukocytosis, elevated lactate dehydrogenase and splenomegaly. In this study, we evaluated the relative frequency of these four criteria in a group of 170 pre-PMF patients and compared them to 225 ET cases. More than 91% of pre-PMF cases showed one or more of these features required for diagnosis, by contrast with only 48% of ET patients. According to clinical data the cumulative risk of progression to advanced/overt PMF in pre-PMF was 36.9% after 15 years. After fitting cox regression models to analyze the impact of the minor criteria on overall survival, only leukocytosis remained as a significant predictor of survival in both pre-PMF and ET. Molecular characterization showed differences in survival in pre-PMF but not ET, with CALR being a more favorable mutation than JAK2. The different outcome of pre-PMF versus ET and associated molecular genetic data supports the concept of two different entities, rather than a continuum of the same disease. Although slightly less than 50% of ET patients also show one or more minor clinical criteria, accurate distinction between ET and pre-PMF is possible by following an integrated approach including histomorphological diagnosis and presence of minor clinical criteria.

Galactomannan testing and Aspergillus PCR in same-day bronchoalveolar lavage and blood samples for diagnosis of invasive aspergillosis.

In recent years galactomannan antigen testing (GM) and also Aspergillus PCR have become increasingly important for diagnosis of invasive aspergillosis (IA). Whether or not these tests need to be performed with bronchoalveolar lavage fluid (BALF; i.e., primary site of infection), or testing of blood samples is sufficient, remains, however, a matter of debate. We evaluated the diagnostic performance of GM ELISA, and Aspergillus PCR by using BALF samples and blood samples obtained at the same day from a total of 53 immunocompromised patients (16 with probable/proven IA and 37 with no evidence of IA according to the revised EORTC/MSG criteria; 38 patients with hematological malignancies were prospectively enrolled at the Medical University of Graz, Austria, 15 patients with mixed underlying diseases at the Mannheim University Hospital). Patients with possible IA were excluded from this analysis. A total of 34/53 (64%) of all patients and 12/16 (75%) of patients with probable/proven IA received mold-active antifungal prophylaxis/therapy at the time of the BALF procedure. Sensitivities of GM and Aspergillus PCR were 38% and 44% in BALF, and 31% and 0% in blood, respectively. Best sensitivity (75%) for detecting proven/probable IA was achieved when BALF Aspergillus PCR, BALF GM (>1.0 ODI), BALF-culture and serum-GM (>0.5 ODI) were combined (specificity 95%). In conclusion, sensitivities of the evaluated diagnostic tests-when interpreted on their own-were low in BALF and even lower in blood, sensitivities increased markedly when diagnostic tests were combined.

Levels of interleukin (IL)-6 and IL-8 are elevated in serum and bronchoalveolar lavage fluid of haematological patients with invasive pulmonary aspergillosis.

Aspergillus spp. have been shown to induce T-helper cell (Th) 1 and Th17 subsets resulting in elevated levels of several cytokines. The objective of this study was to analyse a bundle of cytokines in serum and bronchoalveolar lavage fluid (BALF) in patients with and without invasive pulmonary aspergillosis (IPA). This nested case-control analysis included 10 patients with probable/proven IPA and 20 matched controls without evidence of IPA, out of a pool of prospectively enrolled (2014-2017) adult cases with underlying haematological malignancies and suspected pulmonary infection. Serum samples were collected within 24 hours of BALF sampling. All samples were stored at -70°C for retrospective determination of cytokines. IL-6 and IL-8 were significantly associated with IPA in both serum (P = .011 and P = .028) and BALF (P = .006 and P = .012, respectively), and a trend was observed for serum IL-10 (P = .059). In multivariate conditional logistic regression analysis, IL-10 remained a significant predictor of IPA in serum and IL-8 among BALF cytokines. In conclusion, levels of IL-6 and IL-8 were significantly associated with probable/proven IPA, and a similar trend was observed for serum IL-10. Future cohort studies should determine the diagnostic potential of these cytokines for IPA, and evaluate combinations with other IPA biomarkers/diagnostic tests.

Urine Galactomannan-to-Creatinine Ratio for Detection of Invasive Aspergillosis in Patients with Hematological Malignancies.

Galactomannan (GM) testing of urine specimens may provide important advantages, compared to serum testing, such as easy noninvasive sample collection. We evaluated a total of 632 serial urine samples from 71 patients with underlying hematological malignancies and found that the urine GM/creatinine ratio, i.e., (urine GM level × 100)/urine creatinine level, which takes urine dilution into account, reliably detected invasive aspergillosis and may be a promising diagnostic tool for patients with hematological malignancies. (This study has been registered at ClinicalTrials.gov under registration no. NCT01576653.).

Chemotherapy-Induced Intestinal Mucosal Barrier Damage: a Cause of Falsely Elevated Serum 1,3-Beta-d-Glucan Levels?

Blood citrulline and intestinal fatty acid binding protein were determined as biomarkers for intestinal mucositis. Biomarker levels were correlated with corresponding serum 1,3-beta-D-glucan levels in 56 samples obtained from 33 cases with underlying hematological malignancies receiving induction chemotherapy. No correlation between biomarkers of intestinal mucositis and BDG levels was observed. (This study has been registered at ClinicalTrials.gov under registration no. NCT01576653.).

Therapeutic Resistance in Acute Myeloid Leukemia: The Role of Non-Coding RNAs.

Acute myeloid leukemia (AML) is caused by malignant transformation of hematopoietic stem or progenitor cells and displays the most frequent acute leukemia in adults. Although some patients can be cured with high dose chemotherapy and allogeneic hematopoietic stem cell transplantation, the majority still succumbs to chemoresistant disease. Micro-RNAs (miRNAs) and long non-coding RNAs (lncRNAs) are non-coding RNA fragments and act as key players in the regulation of both physiologic and pathologic gene expression profiles. Aberrant expression of various non-coding RNAs proved to be of seminal importance in the pathogenesis of AML, as well in the development of resistance to chemotherapy. In this review, we discuss the role of miRNAs and lncRNAs with respect to sensitivity and resistance to treatment regimens currently used in AML and provide an outlook on potential therapeutic targets emerging thereof.