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Patients receiving chimeric antigen receptor T cells (CAR-T cells) therapy may be particularly susceptible to coronavirus disease 2019 (COVID-19) because of several factors including the immunosuppression associated to the underlying disease and delayed cytopenias. Regrettably, data on outcomes of CAR-T recipients with COVID-19 are extremely scarce. The aim of this study was to investigate the characteristics and outcomes of COVID-19 in patients treated with CAR-T therapy. The European Hematology Association - Scientific Working Group Infection in Hematology endorsed a survey to collect and analyze data from patients developing COVID-19 after CAR-T therapy. Overall, 459 patients treated with CAR-T cells were reported from 18 European centers. The prevalence of COVID-19 cases was 4.8%. Median time from CAR-T therapy and COVID-19 diagnosis was 169 days. Severe infection occurred in 66.7% of patients and 43.3% of the subjects required admission to ICU. The COVID-19 mortality was 33%. In multivariable analysis, the disease status at the time of COVID-19 trended marginally towards adverse outcome (P=0.075). In conclusion, we documented a high fatality rate for CAR-T patients with COVID-19, supporting the need to design successful interventions to mitigate the risk of infection in this vulnerable group of patients.

Annals of hematology 100(12), 2943-2956 (2021). doi:10.1007/s00277-021-04615-8, Published by Springer, Berlin ; Heidelberg ; New York

To evaluate long-term real-life results of dasatinib therapy among chronic phase chronic myeloid leukemia patients resistant or intolerant to imatinib, we retrospectively analyzed data of 118 patients treated in centers participating in the database INFINITY. With median follow-up of 37 months, estimated 5-year cumulative incidences of complete cytogenetic and major molecular responses were 78% and 68%, respectively. The estimated 5-year probability of overall survival (OS) and event-free survival (EFS) were 86% and 83%, respectively. Both OS and EFS were significantly improved among patients with

Patients with acute myeloid leukemia (AML) are at high risk of mortality from coronavirus disease 2019 (COVID-19). The optimal management of AML patients with COVID-19 has not been established. Our multicenter study included 388 adult AML patients with COVID-19 diagnosis between February 2020 and October 2021. The vast majority were receiving or had received AML treatment in the prior 3 months. COVID-19 was severe in 41.2% and critical in 21.1% of cases. The chemotherapeutic schedule was modified in 174 patients (44.8%), delayed in 68 and permanently discontinued in 106. After a median follow-up of 325 days, 180 patients (46.4%) had died. Death was attributed to COVID-19 (43.3%), AML (26.1%) or to a combination of both (26.7%). Active disease, older age, and treatment discontinuation were associated with death, whereas AML treatment delay was protective. Seventy-nine patients had a simultaneous AML and COVID-19 diagnosis, with an improved survival when AML treatment could be delayed. Patients with COVID-19 diagnosis between January and August 2020 had a significantly lower survival. COVID-19 in AML patients was associated with a high mortality rate and modifications of therapeutic algorithms. The best approach to improve survival was to delay AML treatment.

NPM1 plasmid standards are required for absolute quantification of minimal residual disease in acute myeloid leukemia patients. The standards are usually obtained, next to commercially constructed gene fragments, from transgenic bacteria colonies. However, this procedure is laborious and very time consuming.We have developed a PCR method that speeds up, simplifies, and streamlines the process of preparing NPM1 plasmid standards. The method is based on a combination of three primers, two surrounding the usual NPM1 mutation position and one over the mutation site. With this method, we were able to clearly distinguish plasmids with at least 15 different NPM1 mutations from the wild-type NPM1 plasmid.With the new approach, preparing NPM1 plasmid standards is easier, identifying NPM1-positive colonies is possible in less than a day and moreover, for a lower price than commercially constructed gene fragments.

The care for younger patients with acute myeloid leukaemia (AML) requires fully equipped medical facilities that have routine experience with the management of this aggressive disorder. Two indepen...

Background: Research objective was to detail COVID-19’s natural trajectory in relation to the Czech population’s viral load.Methods: Our prospective detailed daily questionnaire-based telemonitoring study evaluated COVID-19’s impact among 105 outpatients. In accordance with government quarantine requirements, outpatients were divided into a cohort with two negative tests at the end of the disease (40 patients) and a cohort with a new algorithm (65 patients) following a fourteen-day quarantine.Results: Median follow-up differed significantly between the 2 groups (23 days vs. 16 days). Only 6% of patients were asymptomatic during the entire telemonitoring period. 13% of patients were diagnosed asymptomatic, as suspected contacts, yet later developed symptoms, while the remaining 81% were diagnosed on average 6 days following symptom onset. Telemonitoring enabled precise symptom status chronicling. The most frequently reported complaints were fevers, respiratory issues, and anosmia. Six patients were eventually hospitalized for complications detected early after routine telemonitoring. During the extended follow-up (median 181 days), anosmia persisted in 26% of patients. 79% of patients in the new quarantine algorithm cohort reported no symptoms on day 11 compared to just 56% of patients in the two negative test cohort upon first testing negative (median–19 days). The highest viral load occurred within 0-2 days of initial symptom onset. Both the PCR viral load and two consecutive PCR negative sample realizations indicated high interindividual variability with a surprisingly fluctuating pattern among 43% of patients.Conclusions: No definitive COVID-19 symptoms or set of symptoms excepting anosmia (59%) and/or ageusia (47%) were identified. No preexisting medical conditions specifically foreshadowed disease trajectory in a given patient. Without a PCR negativity requirement for quarantine cessation, patients could exhibit fewer symptoms. Our study therefore highlights the urgent need for routine ambulatory patient telemedicine monitoring, early complication detection, intensive mass education connecting disease demeanor with subsequent swift diagnostics, and, notably, the need to reevaluate and modify quarantine regulations for better control of SARS-CoV-2 proliferation.

Lipocalin 2 (LCN2), a proinflammatory mediator, is involved in the pathogenesis of myeloproliferative neoplasms (MPN). Here, we investigated the molecular mechanisms of LCN2 overexpression in MPN. LCN2 mRNA expression was 20-fold upregulated in peripheral blood (PB) mononuclear cells of chronic myeloid leukemia (CML) and myelofibrosis (MF) patients vs. healthy controls. In addition, LCN2 serum levels were significantly increased in polycythemia vera (PV) and MF and positively correlated with JAK2V617F and mutated CALR allele burden and neutrophil counts. Mechanistically, we identified endoplasmic reticulum (ER) stress and the unfolded protein response (UPR) as a main driver of LCN2 expression in BCR-ABL- and JAK2V617F-positive 32D cells. The UPR inducer thapsigargin increased LCN2 expression &gt, 100-fold, and this was not affected by kinase inhibition of BCR-ABL or JAK2V617F. Interestingly, inhibition of the UPR regulators inositol-requiring enzyme 1 (IRE1) and c-Jun N-terminal kinase (JNK) significantly reduced thapsigargin-induced LCN2 RNA and protein expression, and luciferase promoter assays identified nuclear factor kappa B (NF-κB) and CCAAT binding protein (C/EBP) as critical regulators of mLCN2 transcription. In conclusion, the IRE1–JNK-NF-κB–C/EBP axis is a major driver of LCN2 expression in MPN, and targeting UPR and LCN2 may represent a promising novel therapeutic approach in MPN.

Objectives Emerging invasive fungal infections (IFI) have become a notable challenge. Apart from the more frequently described fusariosis, lomentosporiosis, mucormycosis, scedosporiosis, and certain dematiaceae or yeasts, little is known about extremely rare IFI. Methods Extremely rare IFI collected in the FungiScopeⓇ registry were grouped as Dematiaceae, Hypocreales, Saccharomycetales, Eurotiales, Dermatomycetes, Agaricales, and Mucorales. Results Between 2003 and June 2019, 186 extremely rare IFI were documented in FungiScopeⓇ. Dematiaceae (35.5%), Hypocreales (23.1%), Mucorales (11.8%), and Saccharomycetales (11.3%) caused most IFI. Most patients had an underlying malignancy (38.7%) with acute leukemia accounting for 50% of cancers. Dissemination was observed in 26.9% of the patients. Complete or partial clinical response rate was 68.3%, being highest in Eurotiales (82.4%) and in Agaricales (80.0%). Overall mortality rate was 29.3%, ranging from 11.8% in Eurotiales to 50.0% in Mucorales. Conclusions Physicians are confronted with a complex variety of fungal pathogens, for which treatment recommendations are lacking and successful outcome might be incidental. Through an international consortium of physicians and scientists, these cases of extremely rare IFI can be collected to further investigate their epidemiology and eventually identify effective treatment regimens.

Outcomes of the treatment of older patients with acute myeloid leukemia (AML) are unsatisfactory due to a higher incidence of negative patient- and disease-related risk factors connected with aging. Prediction of poor tolerance to aggressive treatment and low response to standard intensive chemotherapy are the main root causes why the treatment decision is challenging. For a long time, negative prognostic factors for treatment outcomes, overall survival, and early death such as the age itself, low-performance status, high-comorbidity burden, adverse cytogenetics, and secondary AML have been known, and they are routinely taken into account during therapeutic balance. In consideration of the risk factors and specific laboratory results, prognostic models have been created. Despite the abovementioned facts, the survival of older patients with AML remains very poor, that holds true even for the intensive therapy. For that reason, there is an increased effort to find a better approach how to select patients who would benefit from intensive treatment without decreasing their quality of life through severe complications with risk of high treatment-related mortality. Based on the results of clinical studies, the geriatric assessment could be the missing step which would help select older patients who are really fit for intensive treatment and who will benefit from it the most. This review focuses on the risk factors that should be taken under advisement when the decision about the treatment is made. With reference to the published information, we propose an algorithm how to identify fit, vulnerable, and frail patients.

Baseline chest computed tomography (BCT) in high-risk hematology patients allows for the early diagnosis of invasive pulmonary aspergillosis (IPA). The distribution of BCT implementation in hematology departments and impact on outcome is unknown. A web-based questionnaire was designed. International scientific bodies were invited. The estimated numbers of annually treated hematology patients, chest imaging timepoints and techniques, IPA rates, and follow-up imaging were assessed. In total, 142 physicians from 43 countries participated. The specialties included infectious diseases (n = 69, 49%), hematology (n = 68, 48%), and others (n = 41, 29%). BCT was performed in 57% (n = 54) of 92 hospitals. Upon the diagnosis of malignancy or admission, 48% and 24% performed BCT, respectively, and X-ray was performed in 48% and 69%, respectively. BCT was more often used in hematopoietic cell transplantation and in relapsed acute leukemia. European centers performed BCT in 59% and non-European centers in 53%. Median estimated IPA rate was 8% and did not differ between BCT (9%, IQR 5&ndash, 15%) and non-BCT centers (7%, 10%) (p = 0.69). Follow-up computed tomography (CT) for IPA was performed in 98% (n = 90) of centers. In high-risk hematology patients, baseline CT is becoming a standard-of-care. Chest X-ray, while inferior, is still widely used. Randomized, controlled trials are needed to investigate the impact of BCT on patient outcome.

Pegylated interferon-α (peg-IFNa) treatment induces molecular responses (MR) in patients with myeloproliferative neoplasms (MPNs), including partial MR (PMR) in 30–40% of patients. Here, we compared the efficacy of IFNa treatment in JAK2V617F- vs. calreticulin (CALR)-mutated cells and investigated the mechanisms of differential response. Retrospective analysis of MPN patients treated with peg-IFNa demonstrated that patients harboring the JAK2V617F mutation were more likely to achieve PMR than those with mutated CALR (p = 0.004), while there was no significant difference in hematological response. In vitro experiments confirmed an upregulation of IFN-stimulated genes in JAK2V617F-positive 32D cells as well as patient samples (peripheral blood mononuclear cells and CD34+ hematopoietic stem cells) compared to their CALR-mutated counterparts, and higher IFNa doses were needed to achieve the same IFNa response in CALR− as in JAK2V617F-mutant 32D cells. Additionally, Janus-activated kinase-1 (JAK1) and signal transducers and activators of transcription 1 (STAT1) showed constitutive phosphorylation in JAK2V617F-mutated but not CALR-mutated cells, indicating priming towards an IFNa response. Moreover, IFN-induced growth arrest was counteracted by selective JAK1 inhibition but enhanced by JAK2 inhibition. In conclusion, our data suggest that, clinically, higher doses of IFNa are needed in CALR-mutated vs. JAK2V617F-positive patients and we suggest a model of JAK2V617F-JAK1/STAT1 crosstalk leading to a priming of JAK2V617F-positive cells to IFNa resulting in differential sensitivity.

Tyrosine kinase inhibitors (TKI) have dramatically improved the prognosis of CML patients with life expectancy close to individuals without CML [1], and therefore the longterm safety of TKIs has become especially important due to the potential for lifelong treatment. Nilotinib (NILO) tends to have a negative effect on lipid [2] and glucose [3] metabolism, while imatinib (IMA) seems to improve [4] these abnormalities. Although the data appear convincing, the real impact of these changes on cardiovascular (CV) risk and the development of CV complications during TKI therapy have not yet been confirmed.

Introduction Coronavirus disease 2019 (COVID-19) is a life-threatening condition of high relevance for co-morbid patients, such as those with baseline hematological malignancies (HM). One year after the diagnosis of the first COVID-19 case, at the end of 2020, the first vaccines against the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) were administered to the population, starting with individuals at highest risk of infection. EPICOVIDEHA aims to describe the epidemiology, vaccination strategies and mortality rates from HM patients at risk. Methods We collected clinical and epidemiological data from patients with laboratory-based diagnosis of SARS-CoV-2 infection after partial or complete vaccination. The study was sponsored by the European Hematology Association - Infectious Diseases Working Party. Patients were registered in the EPICOVIDEHA online survey between January 1, 2021 and July 31, 2021 from Europe and United States. Data captured included underlying conditions prior to SARS-CoV-2, HM status and management prior to SARS-CoV-2, SARS-CoV-2 vaccination and infection details and mortality. The survey will continue until December 31, 2021. Results Overall, 40 patients have been so far registered, 24 male and 16 females, the vast majority of them aged over 50 years (N=38, 95%). Three quarters of patients were affected by lymphoproliferative malignancies (chronic lymphoid leukemia [CLL] N=14 and non-Hodgkin lymphoma [NHL] and multiple myeloma [MM] N=8, each), followed by myelodysplastic syndrome (MDS) (N=4), acute myeloid leukemia (AML) (N=2) and others (chronic myeloid leukemia [CML], acute lymphoid leukemia [ALL], polycythemia vera [PV] and aggressive mastocytosis one of each). Thirty-one patients (77.5%) were receiving active treatment for underlying HM at the time of SARS-CoV-2 infection, with 16 of them being on chemotherapy in the month prior to infection. All patients were vaccinated with a median time from vaccine to SARS-CoV-2 infection of 45.5 days (IRQ 19-67.5). Twenty-nine patients received a mRNA vaccine (BioNTech/Pfizer N=28, Moderna COVE N=1), whereas the remaining 11 an inactivated vaccine (Sinovac CoronaVac N=6) and vector-based vaccine (AstraZeneca Oxford N=5). Twenty-three patients were completely vaccinated, of which 22 (97.5%) patients were immunized (a minimum of 15 days following second dose). On the contrary, among 17 patients partially vaccinated, none was immunized. In 9 cases, viral genomes were analyzed (English variant N=7, South Africa variant N=1, Indian variant N=1). Overall, 25/40 patients presented with a severe/critical infection (62.5%), 13 of which (52%) were fully vaccinated and immunized, whereas only 15 (37.5%) were asymptomatic or mildly symptomatic. Twenty-seven (92.5%) patients were admitted to hospital, 5/27 (18.5%) to ICU, all requiring mechanical ventilation. After a follow-up of 30 day from SARS-CoV-2 infection, 8 patients died (20%), with 7/8 deaths (87.5%) attributable to SARS-CoV-2. There was no difference in overall survival between those patients that received 2 doses of vaccine or 1 dose (figure 1a), as well as no difference being observed between patients with and without lymphoproliferative malignancies (figure 1b), patients that receiving/not receiving active treatment in the last month (figure 1c), or the type of vaccine injected (figure 1d). Conclusions Our survey, involving over 150 Hematology Departments around the world, provides some preliminary insights. The majority of patients who do not respond to vaccination are patients with lymphoproliferative diseases, as can also be observed for other types of vaccination (e.g., flu-vaccination). Dramatically the mortality observed in all patients, although lower than that observed in the pre-vaccination period which in our experience was around 31%, still remains high (20%). Recruitment to this survey continues, and we hope that with larger numbers of cases, more definitive conclusions can be drawn to develop strategies to keep these complex patients safe. Figure 1 Figure 1. Disclosures Lopez-Garcia: Celgene: Other: Speaker Honoraria; Abbvie: Other: Speaker Honoraria, Advisor, Travel and accommodation grants; Janssen: Other: Speaker Honoraria, Advisor, Travel and accommodation grants, Research Funding; Roche: Other: Speaker Honoraria, Travel and accommodation grants; Novonordisk: Other: Speaker Honoraria; Fresenius: Other: Speaker Honoraria. Glenthoej: Novo Nordisk: Honoraria; Agios: Consultancy, Membership on an entity's Board of Directors or advisory committees; Novartis: Consultancy, Membership on an entity's Board of Directors or advisory committees; bluebird bio: Consultancy, Membership on an entity's Board of Directors or advisory committees; Celgene: Consultancy, Membership on an entity's Board of Directors or advisory committees; Alexion: Research Funding. Mikulska: Biotest: Speakers Bureau; Janssen: Speakers Bureau; MSD: Speakers Bureau; Gilead: Speakers Bureau; Pfizer: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau. Busca: Gilead Sciences: Other: Lecture Honoraria; Merck: Other: Lecture Honoraria; Pfizer Pharmaceuticals: Other: Lecture Honoraria; Basilea: Other: Lecture Honoraria; Biotest: Other: Lecture Honoraria; Jazz Pharmaceuticals: Other: Lecture Honoraria; Takeda: Membership on an entity's Board of Directors or advisory committees. Corradini: KiowaKirin; Incyte; Daiichi Sankyo; Janssen; F. Hoffman-La Roche; Kite; Servier: Consultancy; AbbVie, ADC Theraputics, Amgen, Celgene, Daiichi Sankyo, Gilead/Kite, GSK, Incyte, Janssen, KyowaKirin, Nerviano Medical Science, Novartis, Roche, Sanofi, Takeda: Honoraria; AbbVie, ADC Theraputics, Amgen, Celgene, Daiichi Sankyo, Gilead/Kite, GSK, Incyte, Janssen, KyowaKirin, Nerviano Medical Science, Novartis, Roche, Sanofi, Takeda: Consultancy; Amgen; Takeda; AbbVie: Consultancy, Honoraria, Other: Travel and accommodations; Novartis; Gilead; Celgene: Consultancy, Other: Travel and accommodations; BMS: Other: Travel and accommodation; Sanofi: Consultancy, Honoraria; Incyte: Consultancy; Novartis, Janssen, Celgene, BMS, Takeda, Gilead/Kite, Amgen, AbbVie: Other: travel and accomodations. Hoenigl: Gilead, Pfizer, Astellas, Scynexis, and NIH: Research Funding. Klimko: Gilead Science, MSD, Pfizer: Membership on an entity's Board of Directors or advisory committees; Gilead Sciences, MSD, Pfizer Pharmaceuticals, and Astellas Pharma: Speakers Bureau. Pagliuca: Gentium/Jazz Pharmaceuticals: Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Gilead, Pfizer, and MSD: Research Funding. Passamonti: Celgene: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Novartis: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; AbbVie: Speakers Bureau; BMS: Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; Janssen: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau. Köhler: German Federal Ministry of Research and Education and the State of North Rhine-Westphalia, Germany: Other: Support; Miltenyi Biotec GmbH, Bergisch Gladbach, Germany, and the Cologne Excellence Cluster on Cellular Stress Responses in Aging-Associated Diseases, University of Cologne, Cologne, Germany: Other: Non-financial grants; Akademie für Infektionsmedizin e.V., Ambu GmbH, Astellas Pharma, European Confederation of Medical Mycology, Gilead Sciences, GPR Academy Ruesselsheim, MSD Sharp & Dohme GmbH, Noxxon N.V., and University Hospital, LMU Munich: Consultancy, Honoraria. Cornely: Amplyx, Basilea, BMBF, Cidara, DZIF, EU-DG RTD (101037867), F2G, Gilead, Matinas, MedPace, MSD, Mundipharma, Octapharma, Pfizer, Scynexis: Other: Grants or Contracts. Pagano: Gilead Science, MSD, Pfizer, Basilea, Janssen, Novartis, Jazz Pharmaceutical, Cidara: Membership on an entity's Board of Directors or advisory committees; Gilead Sciences, MSD, Pfizer Pharmaceuticals, Astellas Pharma: Speakers Bureau; Menarini: Consultancy.

Background: SARS-CoV-2 infection may be a threat for those undergoing active anti-cancer therapy. We aim to study adverse events, efficacy, and immune response in Covid-19 vaccinated patients focusing on possibly interfering therapy. Methods: CoVigi is a prospective open-label multicentric phase 4 clinical study (EudraCT 2021-000566-14) enrolling patients on anti-cancer treatment. Vaccines from Pfizer-BioNTech, AstraZeneca, Johnson&Johnson, or Moderna are considered. Data on vaccination side effects, the onset and course of Covid-19, and quantitative analysis of anti-S and anti-N SARS-CoV-2 antibodies (Roche) and SARS-CoV-2 specific cellular response evaluated by IFN-gamma-release assay (Qiagen) and CD69 expression are recorded as follows: at the baseline (prior to the vaccination), prior to the 2nd dose, 4–8 weeks, 3, 6 and 12 months after the first dose. Results: The trial was initiated on March 22th. As of May 4th, 152 solid cancer and 103 hematooncology patients were enrolled. From preliminary baseline data, 22% of solid cancer and 29% of hematooncology patients had detectable levels of anti-S antibodies with a median of 106 U/ml (range 1.4–3666) and 84 U/ml (range 0.75–2528), respectively (p = 0.888). Surprisingly, only 44% solid cancer and 53% of hematooncology patients with detectable antibodies prior to the vaccination referred on covid-19 in medical history. In the Ab-positive cohort, the IFN-gamma level upon both CD4 and CD8 stimulation was 0.04 pg/ml (IQR 0.02–0.13), the CD69 expression on NKT-like cells increased to 10.9% (IQR 6.6–17.3), whereas in the Ab-negative cohort was 0.00 pg/ml (IQR 0.00–0.01 and to 7.5% (IQR 4.0–10.1), respectively (p < 0.001 and p = 0.079). Conclusions: Substantial number of cancer patients experienced SARS-CoV-2 infection during active anti-cancer treatment prior to vaccination, often with asymptomatic course. In SARS-CoV-2-immunized patients, we observed SARS-CoV-2 positive cellular response. The preliminary results with dynamics of immune response with 3-month follow-up will be presented at the conference. Acknowledgment: CZECRIN LM2018128, Roche Diagnostics, MMCI00209805, MHCZ/DRO (FNBr, 65269705). Clinical trial identification: EudraCT 2021-000566-14. Legal entity responsible for the study: Masaryk University. Funding: CZECRIN. Disclosure: All authors have declared no conflicts of interest.

Background Advances in the survival of patients with invasive aspergillosis (IA) are jeopardized by the emergence of azole resistance in Aspergillus fumigatus, which has been associated with high probability of azole treatment failure. The clinical implications of azole-resistant IA compared to azole-susceptible IA remain unclear. Thus, we seek to describe the epidemiology and to determine the efficacy of antifungal therapy in patients with documented azole-resistant IA compared to azole-susceptible IA in patients with hematological malignancy. Methods For proven and probable IA (EORTC/MSG 2019) caused by A. fumigatus in patients with hematological malignancies retrospective data were documented, comprising demographics, diagnosis, treatment, response, and outcome. Sites provided susceptibility results or respective isolates for analysis in a central laboratory. Results Sites in 16 countries worldwide enrolled 187 cases diagnosed with IA between 2010 and 2019; 31 (16.6%) were resistant to at least one of the clinical azoles. Fungal isolates were available from 42 cases. A mixed fungal infection was reported for 32 patients (17.1%), most were related to non-fumigatus Aspergillus and non-Aspergillus molds (n=22, 69%). Most patients were male (66.8%) and overall the majority of patients were in the age groups between 50 and 89 years (71%). Amphotericin B was used for treatment in 24 (77%) patients with azole-resistant IA, compared to 76 (49%) in the azole-susceptible group (lipid-based formulation in 98%); only five (16%) patients with azole-resistant IA were treated with an azole alone vs. 57 (36%) of those with azole-susceptible IA. Overall, all-cause mortality rate was higher for patients with azole-resistant compared to azole-susceptible IA (74.2% vs. 53.8%, log rank P=0.004), the 8 patients with an azole-resistant IA treated in the intensive care unit died within 1 month (Figure 1). Details on underlying disease and survival are given in Table 1. Table 1. Underlying hematological malignancy and clinical outcome of patients with azole-resistant and azole-susceptible invasive aspergillosis Figure 1. Intensive care unit 1-year survival probability for patients with azole-resistant and azole-susceptible invasive aspergillosis Conclusion Azole-resistance in IA is associated with worse outcome, especially in critically ill patients. Susceptibility testing should be considered in patients with a suspected azole-resistant IA to support treatment decisions. Disclosures Danila Seidel, PhD, Basilea (Other Financial or Material Support, travel grant) Oliver Cornely, Prof., Actelion (Grant/Research Support)Actelion (Other Financial or Material Support, Personal fees)Al Jazeera Pharmaceuticals (Consultant)Allecra Therapeutics (Other Financial or Material Support, Personal fees)Amplyx (Other Financial or Material Support, Personal fees)Amplyx (Grant/Research Support)Astellas (Grant/Research Support)Astellas (Other Financial or Material Support, Personal fees)Basilea (Other Financial or Material Support, Personal fees)Basilea (Grant/Research Support)Biosys UK Limited (Other Financial or Material Support, Personal fees)Cidara (Other Financial or Material Support, Personal fees)Cidara (Grant/Research Support)Da Volterra (Grant/Research Support)Da Volterra (Other Financial or Material Support, Personal fees)Entasis (Other Financial or Material Support, Personal fees)F2G (Other Financial or Material Support)F2G (Grant/Research Support)Gilead (Grant/Research Support)Gilead (Other Financial or Material Support, Personal fees)Grupo Biotoscana (Other Financial or Material Support, Personal fees)Janssen Pharmaceuticals (Grant/Research Support)Matinas (Other Financial or Material Support, Personal fees)Medicines Company (Grant/Research Support)MedPace (Grant/Research Support)MedPace (Other Financial or Material Support, Personal fees)Melinta Therapeutics (Grant/Research Support)Menarini Ricerche (Other Financial or Material Support, Personal fees)Merck/MSD (Other Financial or Material Support, Personal fees)Merck/MSD (Grant/Research Support)Mylan Pharmaceuticals (Consultant)Nabriva Therapeutics (Other Financial or Material Support, Personal fees)Octapharma (Other Financial or Material Support, Personal fees)Paratek Pharmaceuticals (Other Financial or Material Support, Personal fees)Pfizer (Other Financial or Material Support, Personal fees)Pfizer (Grant/Research Support)PSI (Other Financial or Material Support, Personal fees)Rempex (Other Financial or Material Support, Personal fees)Roche Diagnostics (Other Financial or Material Support, Personal fees)Scynexis (Other Financial or Material Support, Personal fees)Scynexis (Grant/Research Support)Seres Therapeutics (Other Financial or Material Support, Personal fees)Tetraphase (Other Financial or Material Support, Personal fees) Philipp Koehler, MD, Akademie für Infektionsmedizin e.V., (Other Financial or Material Support, Personal fees)Astellas Pharma GmbH (Other Financial or Material Support, Personal fees)Cologne Excellence Cluster on Cellular Stress Responses in Aging-Associated Diseases, University of Cologne, Cologne, Germany (Other Financial or Material Support, Other)Gilead Sciences GmbH (Other Financial or Material Support, Personal fees)GPR Academy Ruesselsheim (Speaker’s Bureau)Miltenyi Biotec GmbH (Other Financial or Material Support, Non-financial support)MSD Sharp & Dohme GmbH (Other Financial or Material Support, Personal fees)Noxxon N.V. (Speaker’s Bureau)University Hospital, LMU Munich (Other Financial or Material Support, Personal fees) Katrien Lagrou, n/a, FUJIFILM WAKO (Speaker’s Bureau)Gilead (Consultant, Speaker’s Bureau)MSD (Consultant, Speaker’s Bureau, Other Financial or Material Support, travel grant)Pfizer (Speaker’s Bureau, travel grant)SMB Laboratoires Brussels (Consultant) Zdenek Racil, n/a, Astellas (Grant/Research Support, Speaker’s Bureau, travel grant) Blandine Rammaert, n/a, Gilead (Speaker’s Bureau, Other Financial or Material Support, travel grant)Merck/MSD (Speaker’s Bureau)Pfizer (Other Financial or Material Support, travel grant) Nikolay Klimko, n/a, Astellas (Speaker’s Bureau)Gilead (Speaker’s Bureau)Merck/MSD (Speaker’s Bureau)Pfizer (Speaker’s Bureau) Sung-Yeon Cho, MD, Gilead (Grant/Research Support, Speaker’s Bureau)Merck Sharp & Dohme (Grant/Research Support, Speaker’s Bureau)Pfizer (Grant/Research Support, Speaker’s Bureau)

Invasive fungal disease represents one of the severe complications in haematopoietic stem cell transplant recipients. We describe a case of a patient treated for relapse of chronic lymphoblastic leukaemia 6 years after HSCT. The patient was treated for invasive pulmonary aspergillosis but died 3 months later from multiple organ failures consisting of haemorrhagic necrotizing fungal pneumonia, refractory chronic hepatic graft versus host disease and cytomegalovirus hepatitis. Autopsy samples revealed histopathological evidence of fungal hyphae and an unusual Aspergillus nidulans-like species was isolated in pure culture. More precise identification was achieved by using scanning electron microscopy of ascospores and sequencing of calmodulin gene, and the isolate was subsequently re-identified as A. sublatus (section Nidulantes) and showed good in vitro susceptibility against all classes of antifungals. Commonly used ITS rDNA region and β-tubulin gene fail to discriminate A. sublatus from related pathogenic species, especially A. quadrilineatus and A. nidulans. Although this is the first case of proven IPA attributed to A. sublatus, we demonstrated that at least some previously reported infections due to A. quadrilineatus were probably caused by this cryptic species.

Despite advances in the treatment of invasive fungal diseases (IFD), mortality rates remain high. Moreover, due to the expanding spectrum of causative agents, fast and accurate pathogen identification is necessary. We designed a panfungal polymerase chain reaction (PCR), which targets the highly variable ITS2 region of rDNA genes and uses high resolution melting analysis (HRM) for subsequent species identification. The sensitivity and specificity of this method was tested on a broad spectrum of the most clinically important fungal pathogens including Aspergillus spp., Candida spp. and mucormycetes. Despite the fact that fluid from bronchoalveolar lavage (BAL) is one of the most frequently tested materials there is a lack of literature sources aimed at panfungal PCR as an IFD diagnostic tool from BAL samples. The applicability of this method in routine practice was evaluated on 104 BAL samples from immunocompromised patients. Due to high ITS region variability, we obtained divergent melting peaks for different fungal species. Thirteen out of 18 patients with proven or probable IFD were positive. Therefore, the sensitivity, specificity, positive predictive value and negative predictive value of our method were 67%, 100%, 100%, and 94%, respectively. In our assay, fungal pathogens identification is based on HRM, therefore omitting the expensive and time consuming sequencing step. With the high specificity, positive and negative predictive values, short time needed to obtain a result, and low price, the presented assay is intended to be used as a quick screening method for patients at risk of IFD.

Little is known about the function and phenotype of leukemic stem cells (LSCs) in chronic myeloid leukemia (CML) or about specific markers that discriminate LSCs from normal hematopoietic stem cells (HSCs). CD26 has recently been described as a specific marker of CML LSCs. In the current study, we investigated this marker in a cohort of 31 unselected CML patients. BCR/ABL1 positivity was analyzed in highly enriched stem cell fractions using fluorescence in situ hybridization (FISH) and reverse transcription PCR (RT-PCR). The proportion of CD26+ LSCs and CD26− HSCs varied considerably among the patients analyzed, and the percentage of CD26+ cells correlated with leukocyte count. The CD26 expression robustly discriminated LSCs from HSCs. This required a strict gating of the stem cell compartment. Thus, in patients with very low LSC or HSC numbers, only the highly sensitive RT-PCR method discriminated between clonal and non-clonal cells, while a robust FISH analysis required larger numbers of cells in both compartments. Finally, our data show that the numbers of CD26+ CML LSCs correlate with responses to treatment with BCR-ABL1 inhibitors.

Rapid diagnostics of fungal pneumonia and initiation of appropriate therapy are still challenging. In this study, we used two panfungal assays to test bronchoalveolar lavage fluid (BALF) samples to prove their ability to confirm invasive fungal disease diagnosis and identify causative agents. Two methods targeting different fungal rDNA regions were used, and the obtained PCR products were sequenced directly or after cloning. In total, 106 BALF samples from 104 patients were tested. After sequencing, we obtained 578 sequences. Four hundred thirty-seven sequences were excluded from further analysis due to duplication (n = 335) or similarity with sequences detected in the extraction control sample (n = 102); 141 unique sequences were analyzed. Altogether, 23/141 (16%) of the fungi detected belonged to pathogenic species, and 63/141 (45%) were identified as various yeasts; a variety of environmental or very rare fungal human pathogens represented 29/141 (21%) of the total and 26/141 (18%) were described as uncultured fungus. Panfungal PCR detected fungal species that would be missed by specific methods in only one case (probable cryptococcosis). Panfungal PCR followed by sequencing has limited use for testing BALF samples due to frequent commensal or environmental fungal species pickup.

Disseminated fusariosis is a life-threatening, invasive, opportunistic infection in immunocompromised patients, especially those with haematological malignancies. The prognosis is poor because these fungi are resistant to many of the available antifungal agents. We present a case of disseminated fusariosis caused by Fusarium proliferatum in a patient with severe aplastic anaemia complicated by a secondary infection of Aspergillus flavus, with a fatal outcome. We also review the documented Fusarium infections in immunocompromised hosts.

BACKGROUND: Antiviral resistance development is a serious complication of human cytomegalovirus virostatic therapy caused by mutations in UL 97 and/or UL54 genes. OBJECTIVES: To determinate the presence of sensitive and resistant strains in patients developing antiviral resistance. STUDY DESIGN: We used three different molecular biological methods for mutation analysis-restriction fragment length polymorphism, sequencing and real-time PCR approach. RESULTS: We describe three allogeneic hematopoietic stem cell transplant patients developing the GCV resistant HCMV strains manifested by virostatic treatment failure. In these patients we identified UL97 mutations L595S, A594V and A594T and monitored the dynamics of coexisted sensitive/resistant strains. We confirmed the presence of mixed HCMV populations and in two patients a phenomenon of sensitive strain repopulation which occurred after 6.5 months and 1 month after removing GCV pressure. CONCLUSIONS: Our results show changes in proportions of sensitive/resistant subpopulations over time but other studies would be required to demonstrate the beneficial impact of their monitoring on clinical outcome.

Rapid differential diagnostics of pulmonary infiltrates suspected of invasive fungal disease in an immunocompromised host and early initiation of effective antifungal therapy are crucial for patient outcomes. There are no serological tests available to detect mucormycetes; therefore, PCR-based methods are highly suitable. We validated our previously published PCR followed by high-resolution melt analysis (PCR/HRMA) to detect Rhizopus spp., Rhizomucor pusillus , Lichtheimia corymbifera , and Mucor spp. in bronchoalveolar lavage (BAL) samples from immunocompromised patients who were at risk of invasive fungal disease. All PCR/HRMA-positive samples were retested using novel real-time quantitative PCR (RQ PCR) assays specific to the species identified. In total, between January 2009 and December 2012 we analyzed 99 BAL samples from 86 patients with pulmonary abnormalities using PCR/HRMA. Ninety (91%) BAL samples were negative, and 9 (9%) samples were positive. The sensitivity and specificity of PCR/HRMA were 100% and 93%, respectively. By combining the positive results of PCR/HRMA with positive RQ PCR results, the specificity was raised to 98%. PCR/HRMA, due to its high negative predictive value (99%), represents a fast and reliable tool for routine BAL sample screening for the differential diagnosis of pulmonary infiltrates in immunocompromised patients for the four most clinically important mucormycetes.

Summary Objectives To evaluate risk factors, diagnostic procedures, and treatment outcomes of invasive aspergillosis (IA) in patients with hematological malignancies. Methods A retrospective analysis of data from proven/probable IA cases that occurred from 2005 to 2009 at 10 hematology centers was performed. Results We identified 176 IA cases that mainly occurred in patients with acute leukemias (58.5%), mostly those on induction/re-induction treatments (39.8%). Prolonged neutropenia was the most frequent risk factor for IA (61.4%). The lungs were the most frequently affected site (93.8%) and computed tomography detected abnormalities in all episodes; however, only 53.7% of patients had findings suggestive of IA. Galactomannan (GM) detection in serum or bronchoalveolar lavage fluid (positive in 79.1% and 78.8% of episodes, respectively) played a crucial role in IA diagnosis. Neutrophil count and antifungal prophylaxis did not influence the GM positivity rate, but empirical therapy decreased this rate (in serum). Of the IA cases, 53.2% responded to initial antifungal therapy. The combination of voriconazole and echinocandin, even as initial or salvage therapy, did not perform better than voriconazole monotherapy ( p =0.924 for initial therapy and p =0.205 for salvage therapy). Neutrophil recovery had a significant role in the response to initial (but not salvage) antifungal therapy. Conclusions Our retrospective analysis identified key diagnostic and treatment characteristics, and this understanding could improve the management of hematological malignancy patients with IA.

The diagnosis of invasive fungal diseases (IFD) based on clinical, radiological, and conventional microbiological findings is not reliable and is often delayed. Non-culture-based methods with higher sensitivity and specificity may reduce diagnostic time and result in decreased IFD morbidity and mortality. These methods are now increasingly used to manage patients at risk of IFD. Among available biomarkers, fungal antigens have been investigated as an aid to early diagnosis and are predominantly used as screening tests to prompt antifungal treatment mainly in patients with hematological malignancies. The revised version of the European Organization for Research and Treatment of Cancer/Mycoses Study Group (EORTC/MSG) consensus definitions includes some of these biological markers (galactomannan, 1,3-beta-D-glucan, cryptococcus antigen).

The objective of this retrospective study was to evaluate results from voriconazole therapeutic drug monitoring (TDM) in haematological patients in routine clinical practice. Between 2005 and 2010, 1228 blood samples were obtained from 264 haematological patients (median 3 samples/patient; range 1-27) receiving voriconazole for targeted/preemptive treatment of invasive aspergillosis (IA) (46.3% of samples), empirical therapy (12.9%) or prophylaxis (40.8%). A high-pressure liquid chromatography assay was used to analyse voriconazole concentrations. Clinical and laboratory data were analysed retrospectively. The median of the detected voriconazole plasma concentration was 1.00 μg ml(-1) (range

Summary Background We evaluated the performance of a galactomannan (GM) assay in bronchoalveolar lavage (BAL) fluid compared to serum samples for the diagnosis of invasive pulmonary aspergillosis (IPA) in patients with hematological diseases. Methods Two hundred and fifty-five bronchoscopies were performed on 230 patients. Bronchial and alveolar samples from BAL fluid as well as serum samples were analyzed in the GM assay. Results Twenty-eight cases of IPA (11%) were diagnosed. The sensitivity, specificity, positive predictive value, and negative predictive value of the GM assay using a cut-off of 0.5 were 57.1%, 99.3%, 94.1%, and 92.5%, respectively, for the alveolar sample; 44.0%, 99.3%, 91.7%, and 91.4%, respectively, for the bronchial sample; and 60.7%, 100%, 100%, and 92.9%, respectively, for serum. The highest sensitivity (78.6%) with good specificity (98.6%) was obtained with a ‘triple detection' of GM in bronchial, alveolar, and serum samples. Neutropenia and antifungal therapy for only 24h increased the sensitivity, while antifungal treatment for ≥2 days decreased assay performance. Moreover, a trend towards a higher volume of aspirated fluid in GM-negative BAL ( p =0.092) was observed. Conclusions In contrast to recently published data, we found only moderate sensitivity, but high specificity and high positive predictive value of the detection of GM in BAL fluid. In addition, neutropenia, antifungal therapy, and BAL standardization affected GM assay performance.

We have evaluated the contribution of the 1,3-β-d-glucan (BG) assay for the screening of invasive fungal infections (IFIs) in patients with haematological malignancies. Serum samples from patients at risk of IFI were collected twice a week and retrospectively tested using the BG assay. BG screening was performed on 1143 samples from 91 patients during 104 anticancer treatment cycles. Proven and probable cases of IFI occurred in 9 (8.7 %) treatment cycles. Depending on the criterion of positivity used (1× >60 pg ml−1, 1× >80 pg ml−1, 2× >60 pg ml−1 or 2× >80 pg ml−1) the sensitivity and specificity were 89, 89, 67 and 44 %, and 20, 48, 33 and 56 %, respectively. Although the test was marked as positive in 82, 68, 54 and 45 % of all the treatment cycles, in the majority of cases, these positivities were probably false. The major limit of the BG test was an extremely low positive predictive value (10 to 12 %). We have analysed mucositis, candida colonization, bacteraemia, use of antimicrobials, erythrocyte and thrombocyte filtered blood products, collecting tubes or sampling via venous catheters. Even though no factor is a major source of BG, it could at least partially influence BG assay performance. Thus, BG detection has a limited usefulness as a screening method for IFIs in patients with haematological malignancies.

We present a method for rapid and simple detection of clinically relevant mucormycetes of the Mucorales order in cultures and clinical samples. This seminested real-time PCR uses mucormycete-specific primers and is followed by species identification using high-resolution melt (HRM) analysis. The method is highly suitable for routine clinical diagnostics.

The study showed no benefit of adding HLA-haploidentical PBSC infusion to intensive chemotherapy over standard treatment in elderly AML patients.