The multidisciplinary approach to eosinophilia.

Eosinophilic granulocytes are normally present in low numbers in the bloodstream. Patients with an increased number of eosinophilic granulocytes in the differential count (eosinophilia) are common and can pose a clinical challenge because conditions with eosinophilia occur in all medical specialties. The diagnostic approach must be guided by a thorough medical history, supported by specific tests to guide individualized treatment. Neoplastic (primary) eosinophilia is identified by one of several unique acquired genetic causes. In contrast, reactive (secondary) eosinophilia is associated with a cytokine stimulus in a specific disease, while idiopathic eosinophilia is a diagnosis by exclusion. Rational treatment is disease-directed in secondary cases and has paved the way for targeted treatment against the driver in primary eosinophilia, whereas idiopathic cases are treated as needed by principles in eosinophilia originating from clonal drivers. The vast majority of patients are diagnosed with secondary eosinophilia and are managed by the relevant specialty-e.g., rheumatology, allergy, dermatology, gastroenterology, pulmonary medicine, hematology, or infectious disease. The overlap in symptoms and the risk of irreversible organ involvement in eosinophilia, irrespective of the cause, warrants that patients without a diagnostic clarification or who do not respond to adequate treatment should be referred to a multidisciplinary function anchored in a hematology department for evaluation. This review presents the pathophysiology, manifestations, differential diagnosis, diagnostic workup, and management of (adult) patients with eosinophilia. The purpose is to place eosinophilia in a clinical context, and therefore justify and inspire the establishment of a multidisciplinary team of experts from diagnostic and clinical specialties at the regional level to support the second opinion. The target patient population requires highly specialized laboratory analysis and therapy and occasionally has severe eosinophil-induced organ dysfunction. An added value of a centralized, clinical function is to serve as a platform for education and research to further improve the management of patients with eosinophilia. Primary and idiopathic eosinophilia are key topics in the review, which also address current research and discusses outstanding issues in the field.

IFN-α with dasatinib broadens the immune repertoire in patients with chronic-phase chronic myeloid leukemia.

In chronic myeloid leukemia (CML), combination therapies with tyrosine kinase inhibitors (TKIs) aim to improve the achievement of deep molecular remission that would allow therapy discontinuation. IFN-α is one promising candidate, as it has long-lasting effects on both malignant and immune cells. In connection with a multicenter clinical trial combining dasatinib with IFN-α in 40 patients with chronic-phase CML (NordCML007, NCT01725204), we performed immune monitoring with single-cell RNA and T cell receptor (TCR) sequencing (n = 4, 12 samples), bulk TCRß sequencing (n = 13, 26 samples), flow cytometry (n = 40, 106 samples), cytokine analyses (n = 17, 80 samples), and ex vivo functional studies (n = 39, 80 samples). Dasatinib drove the immune repertoire toward terminally differentiated NK and CD8+ T cells with dampened functional capabilities. Patients with dasatinib-associated pleural effusions had increased numbers of CD8+ recently activated effector memory T (Temra) cells. In vitro, dasatinib prevented CD3-induced cell death by blocking TCR signaling. The addition of IFN-α reversed the terminally differentiated phenotypes and increased the number of costimulatory intercellular interactions and the number of unique putative epitope-specific TCR clusters. In vitro IFN-α had costimulatory effects on TCR signaling. Our work supports the combination of IFN-α with TKI therapy, as IFN-α broadens the immune repertoire and restores immunological function.

Outcomes and endpoints in clinical trials supporting the marketing authorisation of treatments in paediatric acute lymphoblastic leukaemia.

The improvement in acute lymphoblastic leukaemia (ALL) treatment has led research efforts to focus on the unmet medical needs of an increasingly smaller patient cohort with resistant leukaemia and to develop more-targeted agents. Survival and response rates remain the most-prevalent endpoints in paediatric ALL research, but other intermediate clinical endpoints and molecular biomarkers for efficacy and mid- and long-term safety endpoints are also being investigated. The success of current ALL treatment appears to be driving new paradigms to optimise clinical drug development, while at the same time, regulatory tools in place are supporting meaningful drug development in the area.

A retrospective cohort study of patients with eosinophilia referred to a tertiary centre.

INTRODUCTION: Patients with eosinophilia (an increased number of eosinophilic granulocytes > 0.5 × 108/l in the blood) are encountered in all medical specialties and frequently need thorough workup to identify the eliciting causes and decide whether treatment is indicated. In Denmark, highly specialised centres for eosinophilic diseases or conditions have been established to provide a foundation for the management of complicated cases. Here, we present experiences from such a multidisciplinary centre. METHODS: This was a retrospective study of all patients seen in our tertiary centre for eosinophilia in the 2016-2019 period. RESULTS: Referrals mainly derived from specialised secondary care and to a lesser degree from primary care physicians. Patients were either asymptomatic or exhibited symptoms from up to three organ systems and presented a median eosinophil count of 1.7 × 108/l. Up to eight new clonality analyses or imaging studies per patient were performed after referral. One of these, T-cell receptor analysis, was performed frequently but provided limited information, whereas, e.g., flow cytometry proved more clinically applicable owing to its broader diagnostic range. In total, 51 patients were evaluated and classified as secondary (59%), myeloid neoplasm with PDGFRA rearrangement (2%), idiopathic hypereosinophilic syndrome (31%) and idiopathic hypereosinophilia (8%). CONCLUSION: The value of a multidisciplinary and versatile approach in a highly specialised centre has a positive impact on diagnostic processes as well as on the evaluation of treatment need. FUNDING: none. TRIAL REGISTRATION: not relevant.

Genomic profiling of a randomized trial of interferon-α vs hydroxyurea in MPN reveals mutation-specific responses.

Although somatic mutations influence the pathogenesis, phenotype, and outcome of myeloproliferative neoplasms (MPNs), little is known about their impact on molecular response to cytoreductive treatment. We performed targeted next-generation sequencing (NGS) on 202 pretreatment samples obtained from patients with MPN enrolled in the DALIAH trial (A Study of Low Dose Interferon Alpha Versus Hydroxyurea in Treatment of Chronic Myeloid Neoplasms; #NCT01387763), a randomized controlled phase 3 clinical trial, and 135 samples obtained after 24 months of therapy with recombinant interferon-alpha (IFNα) or hydroxyurea. The primary aim was to evaluate the association between complete clinicohematologic response (CHR) at 24 months and molecular response through sequential assessment of 120 genes using NGS. Among JAK2-mutated patients treated with IFNα, those with CHR had a greater reduction in the JAK2 variant allele frequency (median, 0.29 to 0.07; P < .0001) compared with those not achieving CHR (median, 0.27 to 0.14; P < .0001). In contrast, the CALR variant allele frequency did not significantly decline in those achieving CHR or in those not achieving CHR. Treatment-emergent mutations in DNMT3A were observed more commonly in patients treated with IFNα compared with hydroxyurea (P = .04). Furthermore, treatment-emergent DNMT3A mutations were significantly enriched in IFNα-treated patients not attaining CHR (P = .02). A mutation in TET2, DNMT3A, or ASXL1 was significantly associated with prior stroke (age-adjusted odds ratio, 5.29; 95% confidence interval, 1.59-17.54; P = .007), as was a mutation in TET2 alone (age-adjusted odds ratio, 3.03; 95% confidence interval, 1.03-9.01; P = .044). At 24 months, we found mutation-specific response patterns to IFNα: (1) JAK2- and CALR-mutated MPN exhibited distinct molecular responses; and (2) DNMT3A-mutated clones/subclones emerged on treatment.

Ruxolitinib and interferon-α2 combination therapy for patients with polycythemia vera or myelofibrosis: a phase II study.

We report the final 2-year end-of-study results from the first clinical trial investigating combination treatment with ruxolitinib and low-dose pegylated interferon-α2 (PEG-IFNα2). The study included 32 patients with polycythemia vera and 18 with primary or secondary myelofibrosis; 46 patients were previously intolerant of or refractory to PEGIFNα2. The primary outcome was efficacy, based on hematologic parameters, quality of life measurements, and JAK2 V617F allele burden. We used the 2013 European LeukemiaNet and International Working Group- Myeloproliferative Neoplasms Research and Treatment response criteria, including response in symptoms, splenomegaly, peripheral blood counts, and bone marrow. Of 32 patients with polycythemia vera, ten (31%) achieved a remission which was a complete remission in three (9%) cases. Of 18 patients with myelofibrosis, eight (44%) achieved a remission; five (28%) were complete remissions. The cumulative incidence of peripheral blood count remission was 0.85 and 0.75 for patients with polycythemia vera and myelofibrosis, respectively. The Myeloproliferative Neoplasm Symptom Assessment Form total symptom score decreased from 22 [95% confidence interval (95% CI):, 16-29] at baseline to 15 (95% CI: 10-22) after 2 years. The median JAK2 V617F allele burden decreased from 47% (95% CI: 33-61%) to 12% (95% CI: 6-22%), and 41% of patients achieved a molecular response. The drop-out rate was 6% among patients with polycythemia vera and 32% among those with myelofibrosis. Of 36 patients previously intolerant of PEG-IFNα2, 31 (86%) completed the study, and 24 (67%) of these received PEG-IFNα2 throughout the study. In conclusion, combination treatment improved cell counts, reduced bone marrow cellularity and fibrosis, decreased JAK2 V617F burden, and reduced symptom burden with acceptable toxicity in several patients with polycythemia vera or myelofibrosis. #EudraCT2013-003295-12.

Age-related prevalence and clinical significance of neutropenia - isolated or combined with other cytopenias: Real world data from 373 820 primary care individuals.

Neutropenia (NP), that is, an absolute blood neutrophil count (ANC) <1.5 g/L, accompanies various diseases. However, the clinical significance of NP, detected in routine complete blood cell counts (CBC) in primary care, is poorly characterized. Here, from a primary care resource with ANCs from >370 000 individuals, we identified and followed neutropenic subjects for the next 4 years for novel ICD-10 based diagnoses of viral infections and hematological malignancies (ie, previously identified major outcomes in NP individuals) in Danish nationwide health registers. Risk estimates were assessed for children/adolescents (1-18 years) and adults (19-90 years) in relation to NP severity, and for isolated NP, bi- or pancytopenias. We found that NP was observed in 4.9% of children and in 1.9% of adults. The lower the ANC, the likelier was a diagnosis of viral infections or hematological malignancies established during the ensuing 4 years. Among neutropenic children, unspecified viral infections predominated, followed by mononucleosis (with other cytopenias in only 7% and 25% of the cases, respectively). All NP children with acute leukemia presented with bi- or pancytopenia from start of follow-up. In NP adults, hepatitis, followed by HIV, were the most common infections, and acute myelogenous leukemia (AML) and myelodysplastic syndromes (MDSs) the predominating hematological malignancies. Adult NP patients, subsequently diagnosed with hepatitis, HIV or AML, MDS, were bi- or pancytopenic in 42%, 47%, 90% and 91% of cases, respectively. Thus, presence of NP in even one CBC may be the first sign of a latent viral or hematological disorder requiring careful follow-up.

Association of the blood eosinophil count with end-organ symptoms.

INTRODUCTION: Eosinophilia may cause organ dysfunction, but an exact relation between eosinophil blood counts and adverse outcomes has not been described. The aim of the study is to associate in one model both normal and increased blood eosinophil counts to the subsequent development of common conditions in internal medicine, in which eosinophil granulocytes may play a role for the symptoms. METHODS: From the Copenhagen Primary Care Differential Count (CopDiff) Database, we identified 359,950 individuals with at least one differential cell count (DIFF) during 2000-2007. From these, one DIFF was randomly chosen. From the Danish National Patient Register we ascertained organ damage, within four years following the DIFF. Using multivariable logistic regression, odds ratios were calculated and adjusted for previous eosinophilia, sex, age, year, month, CRP and comorbid conditions. RESULTS: Risks for skin- and respiratory disease were increased from above the median eosinophil count of 0.16 × 109/l and reached a plateau around 1.0 × 109/l. Furthermore, risks of most outcomes also increased when the eosinophil count approached zero. CONCLUSIONS: The observed U-shaped association with a plateau of risks around 1 × 109/l indicates that the risk for symptoms due to eosinophilia do not increase proportionate at higher counts. This study demonstrates for the first time that there is indeed an increased risk below median count of 0.16 × 109/l for an increased risk for the same manifestations. Clinically, it means that a normal or even low count of eosinophils do not rule out a risk for organ affection by eosinophils, and may contribute to explain, why patients may have normal eosinophil counts in e.g. asthma or allergy and still have symptoms from the lungs and skin, most likely explained by the extravasation of eosinophils.

Hydrochloric acid prolongs the lifetime of central venous catheters in haematologic patients with bacteraemia.

INTRODUCTION: Bacteraemia in adult patients undergoing treatment for leukaemia is common and associated with profound morbidity and mortality. Infections related to the use of a central venous catheter (CVC) are difficult to eliminate with systemic antibiotics. Premature catheter removal is often due to retained biofilm infection. This study investigated if the additional use of hydrochloric acid (HCl) as an intraluminal lock solution may prolong the lifetime of the CVC. METHODS: The study was performed retrospectively based on a database including patients with a tunnelled Leonard 10 F dual or triple lumen CVC implanted who received HCl instillation due to bacteraemia during a five-year period. RESULTS: In a total of 71 cases of bacteraemia, HCl instil-lation was performed. Following HCI instillation, the CVC was not removed due to infection in 49 out of 71 patients (69%). Furthermore, 22 patients (31%) retained their CVC until the end of treatment. Non-infectious mortality (19/71), accidental pull (2/71) or mechanical CVC dysfunction (6/71) were other reasons for premature removal. Twenty-two catheters (31%) had to be removed due to ongoing infection. The median time from CVC placement until HCl instillation was 39 days. The median time from HCl instillation until removal of CVC was 58 days. The most common bacteriological findings were coagulase-negative staphylococci 34%, Enterococcus spp 14% and Escherichia coli 14%. CONCLUSIONS: The study's findings indicate that a prolonged use of CVC is possible following HCl instillation in adult haematologic patients with bacteraemia. FUNDING: none. TRIAL REGISTRATION: not relevant.

Apheresis principles in a patient with chronic myeloid leukemia during pregnancy: challenges in cell separation and assessing transcript levels.

BACKGROUND: Chronic myeloid leukemia (CML) is rarely diagnosed in pregnant women. CASE REPORT: We report a case of a pregnant woman who presented with a leukocyte count of 250 × 109 cells/L at gestational age (GA) 26 weeks and was diagnosed with CML in the chronic phase. Because the patient deliberately opted out of interferon α and tyrosine kinase inhibitor treatment, the main goal was to reduce the leukocyte count to postpone delivery beyond the number of weeks considered severely premature and avoid thromboembolic complications while continuously evaluating the clinical safety of the mother and fetus. Hence therapeutic leukapheresis was initiated, and we report the first application of an apheresis approach for this procedure using the Spectra Optia instrument without sedimentation agents. Leukapheresis was conducted 2 to 4 times per week for 9 weeks. RESULTS: During treatment the leukocyte count decreased remarkably, and the patient developed lymphopenia together with a paradoxical increase in her blood platelet count. Premature labor was induced at GA 35 weeks, and a healthy boy was delivered. Thereafter, the patient initiated imatinib treatment and was in major molecular and complete cytogenetic remission after 1 year. Despite the remarkable reduction of the leukocyte count, we observed a pronounced increase in expression of BCR-ABL1 transcripts, implying the need for close monitoring of patients with CML during pregnancy. CONCLUSIONS: We report a pregnant woman who was diagnosed with CML and treated solely with apheresis procedures using the Spectra Optia instrument for 9 weeks, ensuring the safe delivery of her child.

Publisher Correction: Epigenetic changes in myelofibrosis: Distinct methylation changes in the myeloid compartments and in cases with ASXL1 mutations.

A correction to this article has been published and is linked from the HTML and PDF versions of this paper. The error has been fixed in the paper.

[Eosinophilia].

When the number of eosinophil granulocytes in blood increases, the cause is not always easy to disentangle. This review highlights the symptoms of rare clonal and common reactive diagnoses, how to approach the patient clinically, and how to implement the armamentarium of available tests in order to identify the correct diagnosis and offer the proper treatment. Two referral centres for eosinophilia have been established in Denmark to support this activity by a collaboration between all departments of haematology and the relevant specialities, meeting the manifestations of eosinophilia.

Safety and efficacy of combination therapy of interferon-α2 and ruxolitinib in polycythemia vera and myelofibrosis.

Interferon-α2 reduces elevated blood cell counts and splenomegaly in patients with myeloproliferative neoplasms (MPN) and may restore polyclonal hematopoiesis. Its use is limited by inflammation-mediated toxicity, leading to treatment discontinuation in 10-30% of patients. Ruxolitinib, a potent anti-inflammatory agent, has demonstrated benefit in myelofibrosis (MF) and polycythemia vera (PV) patients. Combination therapy (CT) with these two agents may be more efficacious than monotherapy with either, potentially improving tolerability of interferon-α2 as well. We report the preliminary results from a phase II study of CT with pegylated interferon-α2 and ruxolitinib in 50 MPN patients (PV, n = 32; low-/intermediate-1-risk MF, n = 18), the majority (n = 47) being resistant and/or intolerant to interferon-α2 monotherapy. Objectives included remission (2013 revised criteria encompassing histologic, hematologic, and clinical responses), complete hematologic response (CHR), molecular response, and toxicity. Follow-up was 12 months. Partial remission (PR) and sustained CHR were achieved in 9% and 44% of PV patients, respectively. In MF patients, complete or partial remission was achieved in 39%, and sustained CHR in 58%. The median JAK2V617F allele burden declined significantly in both groups. Hematologic toxicity was the most common adverse event and was managed by dose reduction. Thirty-seven serious adverse events were recorded in 23 patients; the discontinuation rate was 20%. We conclude that CT with interferon-α2 and ruxolitinib is efficacious in patients with low-/intermediate-1-risk MF and, to a lesser extent, in patients with PV. These preliminary results encourage phase III studies as well as a study with CT in newly diagnosed MPN patients.

Myeloproliferative Neoplasms in Danish Twins.

OBJECTIVE: Myeloproliferative neoplasms (MPNs) are a heterogeneous group of diseases characterized by clonal hyperproliferation of immature and mature cells of the myeloid lineage. Genetic differences have been proposed to play a role in the development of MPNs. Monozygotic twin pairs with MPNs have been reported in a few case reports, but the MPN concordance pattern in twins remains unknown. METHOD: All twin pairs born in the period 1900-2010 were identified in the nationwide Danish Twin Registry. Only pairs with both twins alive on January 1, 1977, and those born thereafter were included to allow identification in the Danish National Patient Registry. RESULTS: A total of 158 twin pairs were registered with an MPN diagnosis: 36 monozygotic, 104 dizygotic, and 18 pairs with unknown zygosity. MPNs were diagnosed in both twins in 4 pairs. The probandwise concordance rates for monozygotic twin pairs were higher than for dizygotic twin pairs (15 vs. 0%; p = 0.016). CONCLUSION: An estimated concordance rate of 15% (95% CI 0.059-0.31) is modest, but given the rarity of MPNs this finding is clinically relevant and provides further support for the role of genetic predisposition in the development of MPNs.

Thromboembolism prophylaxis in patients with Philadelphia-negative myeloproliferative neoplasms-Clinical practice among Nordic specialists.

BACKGROUND: Patients with Philadelphia chromosome-negative myeloproliferative neoplasms (MPNs) have higher risks of developing thromboembolisms compared to the general population. International guidelines on the management of MPNs therefore include recommendations concerning thromboembolism prophylaxis. In clinical practice, strict adherence to guidelines may be challenging and dependent on factors such as physician experience, outpatient clinic setting, and access to therapy; however, no data exist on physician adherence or patient compliance to thromboembolism prophylaxis in MPNs. OBJECTIVES: The Nordic Myeloproliferative Neoplasm Study Group (NMPN) performed a survey among Nordic hematology specialists with the aim of documenting the implementation of international recommendations in a region of Northern Europe with similar healthcare systems. RESULTS: The study showed that Nordic specialists managed their patients in accordance with international guidelines concerning medical intervention, but to a lesser degree regarding the management of additional cardiovascular risk factors. The survey also drew attention to the common clinical dilemma of combining antiaggregatory agents with vitamin K antagonists (VKA), or novel oral anticoagulants (NOAC), as well as phlebotomy limits in female polycythemia vera patients. CONCLUSIONS: The results of this study highlight the importance of considering all risk factors for thrombosis and an optimal collaboration with the primary healthcare sector.

Epigenetic changes in myelofibrosis: Distinct methylation changes in the myeloid compartments and in cases with ASXL1 mutations.

This is the first study to compare genome-wide DNA methylation profiles of sorted blood cells from myelofibrosis (MF) patients and healthy controls. We found that differentially methylated CpG sites located to genes involved in 'cancer' and 'embryonic development' in MF CD34+ cells, in 'inflammatory disease' in MF mononuclear cells, and in 'immunological diseases' in MF granulocytes. Only few differentially methylated CpG sites were common among the three cell populations. Mutations in the epigenetic regulators ASXL1 (47%) and TET2 (20%) were not associated with a specific DNA methylation pattern using an unsupervised approach. However, in a supervised analysis of ASXL1 mutated versus wild-type cases, differentially methylated CpG sites were enriched in regions marked by histone H3K4me1, histone H3K27me3, and the bivalent histone mark H3K27me3 + H3K4me3 in human CD34+ cells. Hypermethylation of selected CpG sites was confirmed in a separate validation cohort of 30 MF patients by pyrosequencing. Altogether, we show that individual MF cell populations have distinct differentially methylated genes relative to their normal counterparts, which likely contribute to the phenotypic characteristics of MF. Furthermore, differentially methylated CpG sites in ASXL1 mutated MF cases are found in regulatory regions that could be associated with aberrant gene expression of ASXL1 target genes.

Non-invasive imaging of retinal blood flow in myeloproliferative neoplasms.

PURPOSE: To study the circulation in the retinal vessels in patients with blood dyscrasia due to myeloproliferative neoplasms using non-invasive retinal imaging. METHODS: Prospective consecutive case series of seven treatment-naïve patients with chronic myeloid leukaemia (n = 2), polycythemia vera (n = 4), essential thrombocytosis (n = 1) examined before and after cytoreductive treatment. We investigated retinal circulation with motion-contrast imaging, retinal oximetry and spectral-domain optical coherence tomography. RESULTS: Retinal venous blood velocity increased by 8.14% (CI95 3.67% to 12.6%, p = 0.004) and retinal arterial oxygen saturation increased by 7.23% (CI95 2.9% to 11.6%, p = 0.010) at follow-up (mean 12 weeks, range 5-14 weeks) where complete haematological remission had been achieved by cytoreductive treatment. Abnormal optical coherence tomography reflectivity patterns were present at baseline in patients with chronic myeloid leukaemia and were replaced by normal patterns at follow-up. Retinopathy, in the form of cotton-wool spots and retinal haemorrhages, was found at presentation in the two patients with chronic myeloid leukaemia and in one patient with polycythemia vera. The retinopathy had resolved at follow-up in all patients. CONCLUSION: With non-invasive retinal imaging, we were able to demonstrate increased retinal venous blood velocity, increased retinal arterial blood oxygenation and normalization of intravascular reflectivity patterns after successful treatment of myeloproliferative neoplasms. Larger prospective studies are needed to assess the prognostic value of these non-invasive imaging methods in predicting circulatory complications in myeloproliferative neoplasms.