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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.

Simple Summary Patients with simultaneous diagnosis of haematological malignancies (HM) and COVID-19 are an even greater challenge for hematologists. To better clarify their outcome, we describe the clinical features and outcome of a cohort of 450 patients with simultaneous diagnosis of HM and COVID-19 registered in the EPICOVIDEHA registry between March 2020 to February 2022. Overall, 343 (76.2%) patients received treatment for HM, and an overall response rate was observed in 140 (40.8%) patients after the first line of treatment. Thirty-day mortality was significantly higher in patients not receiving HM treatment (42.1%) than in those receiving treatment (27.4%, p = 0.004). Statistical analysis showed that, together with age, severe/critical COVID-19, >= 2 comorbidities, lack of HM treatment was an independent risk factors for mortality. These observations suggest the importance of HM treatment in these patients; therefore, it should be delivered as soon as possible for patients requiring immediate therapy. Background: The outcome of patients with simultaneous diagnosis of haematological malignancies (HM) and COVID-19 is unknown and there are no specific treatment guidelines. Methods: We describe the clinical features and outcome of a cohort of 450 patients with simultaneous diagnosis of HM and COVID-19 registered in the EPICOVIDEHA registry between March 2020 to February 2022. Results: Acute leukaemia and lymphoma were the most frequent HM (35.8% and 35.1%, respectively). Overall, 343 (76.2%) patients received treatment for HM, which was delayed for longer than one month since diagnosis in 57 (16.6%). An overall response rate was observed in 140 (40.8%) patients after the first line of treatment. After a median follow-up of 35 days, overall mortality was 177/450 (39.3%); 30-day mortality was significantly higher in patients not receiving HM treatment (42.1%) than in those receiving treatment (27.4%, p = 0.004), either before and/or after COVID-19, or compared to patients receiving HM treatment at least after COVID-19 (15.2%, p < 0.001). Age, severe/critical COVID-19, >= 2 comorbidities, and lack of HM treatment were independent risk factors for mortality, whereas a lymphocyte count >500/mcl at COVID-19 onset was protective. Conclusions: HM treatment should be delivered as soon as possible for patients with simultaneous diagnosis of COVID-19 and HM requiring immediate therapy.

Patients with lymphoproliferative diseases (LPD) are vulnerable to severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection. Here, we describe and analyze the outcome of 366 adult patients with chronic lymphocytic leukemia (CLL) or non-Hodgkin Lymphoma (NHL) treated with targeted drugs and laboratory-confirmed COVID-19 diagnosed between February 2020 and January 2022. Median follow-up was 70.5 days (IQR 0-609). Most used targeted drugs were Bruton-kinase inhibitors (BKIs) (N= 201, 55%), anti-CD20 other than rituximab (N=61, 16%), BCL2 inhibitors (N=33, 9%) and lenalidomide (N=28, 8%).Only 16.2% of the patients were vaccinated with 2 or more doses of vaccine at the onset of COVID-19. Mortality was 24% (89/366) on day 30 and 36%(134/366) on the last day of follow-up. Age >75 years (p, EPICOVIDEHA has received funds from Optics COMMITTM (COVID-19 Unmet Medical Needs and Associated Research Extension) COVID-19 RFP program by GILEAD Science, United States (Project 2020-8223).

All-trans retinoic acid (ATRA)-based therapy for acute promyelocytic leukemia (APL), a subtype of acute myeloid leukemia (AML), is the most successful example of differentiation therapy. Although ATRA can induce differentiation in some non-APL AML cell lines and primary blasts, clinical results of adding ATRA to standard therapy in non-APL AML patients have been inconsistent, probably due to use of different regimens and lack of diagnostic tools for identifying which patients may be sensitive to ATRA. In this study, we exposed primary blasts obtained from non- APL AML patients to ATRA to test for differentiation potential in vitro. We observed increased expression of differentiation markers, indicating a response to ATRA, in four out of fifteen primary AML samples. Three samples in which CD11b increased in response to ATRA had an inversion of chromosome 16 as well as the CBFB- MYH11 fusion gene, and the fourth sample was from a patient with KMT2A-rearranged, therapy-related AML. In conclusion, we identified a subgroup of non- APL AML patients with inv(16) and CBFB-MYH11 as the most sensitive to ATRA-mediated differentiation in vitro, and our results can help identify patients who may benefit from ATRA treatment.

Background All-trans retinoic acid (ATRA)-based treatment of acute promyelocytic leukemia (APL) is the most successful pharmacological treatment of acute myeloid leukemia (AML). Recent development of inhibitors of mutated isocitrate dehydrogenase and dihydroorotate dehydrogenase (DHODH) has revived interest in differentiation therapy of non-APL AML. Our previous studies demonstrated that 5-aminoimidazole-4-carboxamide ribonucleoside (AICAr) induced differentiation of monocytic cell lines by activating the ATR/Chk1 via pyrimidine depletion. In the present study, the effects of AICAr on the viability and differentiation of primary AML blasts isolated from bone marrow of patients with non-APL AML were tested and compared with the effects of DHODH inhibitor brequinar and ATRA. Methods Bone marrow samples were obtained from 35 patients and leukemia blasts were cultured ex vivo. The cell viability was assessed by MTT assay and AML cell differentiation was determined by flow cytometry and morphological analyses. RNA sequencing and partial data analysis were conducted using ClusterProfiler package. Statistical analysis was performed using GraphPad Prism 6.0. Results AICAr is capable of triggering differentiation in samples of bone marrow blasts cultured ex vivo that were resistant to ATRA. AICAr-induced differentiation correlates with proliferation and sensitivity to DHODH inhibition. RNA-seq data obtained in primary AML blasts confirmed that AICAr treatment induced downregulation of pyrimidine metabolism pathways together with an upregulation of gene set involved in hematopoietic cell lineage. Conclusion AICAr induces differentiation in a subset of primary non-APL AML blasts, and these effects correlate with sensitivity to a well-known, potent DHODH inhibitor.

The aim of the current study was to determine whether plasmapheresis in combination with chemotherapy could significantly remove free light chains (FLC) in multiple myeloma (MM) patients with acute kidney injury (AKI) and therefore improve renal recovery and patient survival. During the study period, 29 patients with MM and AKI presented to our unit and were treated with two different therapy modalities (plasmapheresis with chemotherapy or bortezomib). At the end of treatment, a significant decrease of FLCs was present in the group treated with plasmapheresis compared to the bortezomib group. Patients treated with plasmapheresis had similar survival compared to patients treated with bortezomib. There was a significantly higher decrease of FLCs and longer survival in patients treated with three or more plasmapheresis sessions than in patients treated with two plasmapheresis sessions. Plasmapheresis therapy still remains a useful and effective method in the treatment of AKI in MM patients. Plasmapheresis significantly reduces FLCs compared to bortezomib especially with higher number of plasma exchange sessions but it must be combined with other chemotherapy agents in order to prolong renal recovery and therefore patient survival.

This multicenter retrospective study investigated serum calprotectin levels in patients with essential thrombocythemia (ET), polycythemia vera (PV) and myelofibrosis (MF). Serum calprotectin levels were higher in ET, PV, and MF patients when compared to healthy controls ; however, there was no difference in serum calprotectin levels between the three disorders. Higher serum calprotectin levels correlated with clinical and laboratory parameters indicative of a higher systemic inflammation. These results suggested that serum calprotectin may be a novel circulatory inflammatory biomarker in patients with myeloproliferative neoplasms.

Acute lymphoblastic leukemia (ALL) is a malignant disease of lymphoid precursors. According to immunophenotype, it is further subdivided into precursor B cell ALL and precursor T cell ALL, with precursor B cell ALL being much more common both in children and adults. Lineage switch from one lymphoid lineage to another during the course of the disease is extremely rarely reported. Here, we describe a case of a child who initially presented as a precursor B-ALL but 15 years later and after two successfully treated relapses of the original ALL presented with early T cell precursor leukemia. Although it was considered as a relapse, it could be interpreted as a case of secondary leukemia, which can be explained as a consequence of treatment as well as a constitutional feature of an individual. Also, it draws attention to the possibility that hematopoietic cells, and in that context also leukemic cells, are much more plastic and capable of reprogramming than previously thought.

Haemoglobinopathies are hereditary disorders of globin chain synthesis and are the most common inherited diseases worldwide. Haemoglobin E is a structural haemoglobin variant characteristic for South East Asian population. We present a rare and unusual finding of haemoglobin E detected in University Hospital Centre Zagreb by capillary zone electrophoresis. Detection of haemoglobin structural variant helped to avoid misdiagnosis of sideropenic anemia and thus potentially harmful therapeutic intervention. In today’s European multiethnic population haemoglobinopathies are a public health issue and Croatian laboratory professionals should be aware of a possibility of finding an unusual haemoglobin pattern.

AIM: To evaluate the clinical utility of incorporating a novel heavy/light chain immunoassay (HLC) into the existing methods for the assessment of multiple myeloma (MM) patients. ----- METHODS: Convenience sera samples from 90 previously treated IgG and IgA MM patients in different disease stages were analyzed. The study was conducted in Clinical Hospital Center Zagreb between 2011 and 2013. The collected sera were analyzed by standard laboratory techniques (serum protein electrophoresis, quantification of total immunoglobulins, serum immunofixation, serum free light chain [FLC] assay) and HLC assay. ----- RESULTS: HLC ratios outside the normal range were found in 58 of 90 patients, including 28 out of 61 patients with total immunoglobulin measurements within the normal range and 5 out of 23 patients in complete response. Both elevated HLC isotype level and abnormal HLC ratio correlated with the parameters of tumor burden, including percentage of plasma cells in the bone marrow (P3.5mg/L were independent risk factors for survival. ----- CONCLUSION: The new HLC assay has greater sensitivity in detecting monoclonal protein, correlates with tumor burden markers, and affects patients' outcome.

Acute myeloid leukemia (AML) is a malignant hematopoietic disease with poor clinical course and outcome. There is a constant need for new prognostic factors that could facilitate patient risk stratification. The aim of our research was to determine the phosphorylation levels of phosphatidylinositol 3-kinase (PI3K)/Akt and mitogen-activated protein kinase (MAPK) pathways in leukemic cells, their relation to P-glycoprotein (P-gp) expression/activity and their prognostic significance in adult de novo AML. A total of 118 patients with AML were enrolled in the study. In a multivariate Cox regression analysis we found that P-gp activity and Akt phosphorylation were independent poor prognostic factors of overall survival (OS). In contrast, phosphorylated extracellular signal- regulated kinase 1/2 (ERK1/2) represented a favorable prognostic factor of OS and relapse- free survival (RFS). A negative correlation between P-gp activity and p38 phosphorylation level was found, implying a possible role of this MAPK pathway in P-gp regulation. In addition, we found correlation between Akt and p38 phosphorylation levels, indicative of co- activation of two signaling cascades in AML.

Introduction. It has been rarely reported that a laboratory test introduced so rapid and radical changes in diagnostic algorithm as is the case with the quantitative assay of free light chains of immunoglobulins (FLC) in serum and its role in the diagnostic algorithm of monoclonal gammopathies. Since the first description of immunoassay in year 2001 until today, new evidence has continuously been reported in the literature that confirm the clinical usefulness of this test in diagnosis, monitoring and prognosis of monoclonal plasma-proliferative diseases, especially diseases of light chains such as primary amyloidosis, light chain deposition disease (LCDD) or light chain multiple myeloma (LCMM) and nonsecretory multiple myeloma (NSMM). Recently, a commercial test has become available on the market that uses polyclonal antibodies to specific epitopes of free light chains which are hidden in the intact immunoglobulin molecules. In 2011, a commercial immunoassay was launched on the market that uses monoclonal instead of polyclonal antibodies, reducing the variability between different series of reagents and controlling excess antigen in the sample. Aim. The aim of this study was to evaluate monoclonal versus polyclonal antibody and immunoturbidimetric versus immunonephelometric detection technology. Does different detection tehnology – besides different used antibody – contribute to greater variability in results? Method. In this study we compared results of 40 samples measured with polyclonal antibody (The Binding Site Ltd., Birmingham, UK) and monoclonal antibody (N Latex FLC, Siemens Healthcare Diagnostics, Marburg, Germany). In other 40 samples we compared results achieved with different antibodies and different analytical platforms (Siemens Nephelometer with Roche Cobas). Results were statistically analyzed using MedCalc software. Results. Results are shown in Table 1. Comparing all results, it is evident that there is at least proportional error when comparing different antibodies and different analytical systems. Although it is known that immunoturbidimetry is less sensitive than immunonephelometric method, greater discrepancies in results were not found. When we categorized patients as positive and negative according to manufacturer's reference interval for kf/lf ratio, agreement between groups with different antibody and same detection technology was 63% (weighted kappa 0.30). Agreement between groups with different antibodies and different detection technology was 86% (weighted kappa 0.22). Although we have not measured the same samples when testing antibody and analytical platform, the selected analytical platform has, according to our results, no additional impact on the variability of results. Abstract 5680. Table 1. Comparison of FLC results using different antibody and different analytical platforms Method FLC kappa polyclonal Ab Nephelometer Siemens FLC kappa monoclonal Ab Nephelometer Siemens FLC lambda polyclonal Ab Nephelometer Siemens FLC lambda monoclonal Ab Nephelometer Siemens Results (min-max) 6.59-5210.00 6.34-1600.00 1.67-3010.00 1.00-1600.00 Passing-Bablok fit intercept (95% Cl) 8.2442.9255 to 14.9249 1.0945-1.5910 to 5.5631 slope (95% Cl) 0.5950.4564 to 0.7852 1.87981.5336 to 2.1045 Correlation rs (p Conclusion. Physicians and especially clinical biochemists must be aware of the technical shortcomings of this test, such as the variability between different series (lots) reagents, non-linearity, unreliable detection of excess antigen and overestimation of FLC concentrations due to nonspecific interference or polymerization. Although initial results are not discouraging, it will be necessary to collect much more evidence, especially bearing in mind that use of monoclonal antibodies along with advantages has certain disadvantages. In the future, it will probably be necessary to incorporate into the guidelines a recommendation to report the method used, like for other tumor markers. Figure 1 Figure 1. Figure 2 Figure 2. Disclosures No relevant conflicts of interest to declare.

Objectives: To evaluate the clinical utility of the novel heavy/light chain immunoassay for the assessment of multiple myeloma patients. Methods: Serum samples of 99 multiple myeloma patients in different disease stages (74 IgG, 25 IgA) were analysed. Disease assessement was performed by standard laboratory procedures (SPE, serum IFE, serum free light chain assay [FLC], quantitative immunoglobulins, serum beta-2-microglobulin, as well as basic biochemistry and hematology tests) in addition to bone marrow analysis and the novel heavy/light chain assay (HLC). Serum HLC and FLC measurements were obtained using the polyclonal antisera assay Hevylite™ and Freelite™ respectively (The Binding Site, Birmingham, UK) and performed on a Dade BNII analyser (Siemens). SPE and sIFE were performed on a SEBIA Hydrasis platform. Total immunglobulins were measured using Tina-quant Gen.2 reagents on a Roche Cobas 6000cee platform. Statistical analysis was performed using the R package software (R Development Core Team, Vienna, Austria). Results: The majority of patients (61/99) had total immunoglobulin measurements within normal ranges. However, HLC measurement revealed 16/61 patients to have an abnormal HLC ratio, signifying monoclonality. Furthermore, 5/23 patients who had achieved a complete response (CR) were found to have an abnormal HLC ratio, indicating the presence of residual disease. Subsequently, 2/5 of these patients suffered relapse during the follow-up period. Analysing the entire group of patients, notable statistically significant correlations were observed for the percentage of clonal plasma cells in the bone marrow and an abnormal serum FLC ratio with an abnormal HLC ratio (p=0.0004 and p=0.0001 respectively) as well as an extremely abnormal HLC ratio (40; p=0.003 and p=0.01 respectively). Kaplan-Meier analysis for the entire group showed that overall survival (OS) in patients with abnormal HLC ratios or, in particular, extremely abnormal HLC ratio values was significantly shorter (median survival 31.7 month vs. median not reached, p = 0.021; median 11.8 month vs. 31.7 months; p = 0.0032, respectively, figures 1a and 1b). A significantly shorter time to progression (TTP) was also associated with abnormal or extremely abnormal HLC ratio values (median 21.1 months vs median not reached, p = 0.006; median 11.8 months vs 23.1 months, p = 0.0004; figures 2a and 2b). Interestingly, suppression of the polyclonal isotype pair was also associated with a significantly shorter TTP (median 21.1 months vs median not reached, p = 0.029). In multivariate analysis, abnormal HLC ratio values proved to be an independent prognostic risk factor (p = 0.05; table 1). Conclusion: The novel heavy/light chain allows accurate measurement of monoclonal immunoglobulins and can be more sensitive than standard techniques at detecting residual disease. Abnormal heavy/light chain ratios and immunoglobulin isotype pair suppression may have prognostic significance for multiple myeloma patients. Figure 1. Overall survival in patients stratified according to HLC ratio values. a) Median survival was 31.7 months in patients with abnormal HLC ratio values (dotted line) and was not reached in patients with normal values (full line, p = 0.021). b) the difference is even bigger when patients were stratified by extreme values of HLC ratio (< 0.02 or > 40). Median survival was 11.8 months in patients with extreme HLC ratio values (dotted line) and 31.7 months in patients with less extreme values (full line, p = 0.0032). Figure 1. Overall survival in patients stratified according to HLC ratio values. a) Median survival was 31.7 months in patients with abnormal HLC ratio values (dotted line) and was not reached in patients with normal values (full line, p = 0.021). b) the difference is even bigger when patients were stratified by extreme values of HLC ratio (< 0.02 or > 40). Median survival was 11.8 months in patients with extreme HLC ratio values (dotted line) and 31.7 months in patients with less extreme values (full line, p = 0.0032). Figure 2. Time to progression in patients stratified according to HLC ratio values. a) Median time to progression was 21.1 months in patients with abnormal HLC ratio values (dotted line) and was not reached in patients with normal values (full line, p = 0.006). b) when patients were stratified by extreme values of HLC ratio (< 0.02 or > 40), median time to progression was 11.8 months in patients with extreme HLC ratio values (dotted line) and 23.1 months in patients with less extreme values (full line, p = 0.0004). Figure 2. Time to progression in patients stratified according to HLC ratio values. a) Median time to progression was 21.1 months in patients with abnormal HLC ratio values (dotted line) and was not reached in patients with normal values (full line, p = 0.006). b) when patients were stratified by extreme values of HLC ratio (< 0.02 or > 40), median time to progression was 11.8 months in patients with extreme HLC ratio values (dotted line) and 23.1 months in patients with less extreme values (full line, p = 0.0004). Table 1. Multivariate Cox regression analysis Risk factor Relative risk 95% confidence interval "p" value abnormal IgGκ/IgGλ;IgAκ/IgAλ ratio 3.55 0.96 – 13.20 0.05 HLC isotype absolute value > median 0.73 0.27 – 1.97 0.54 abnormal serum FLC ratio 1.24 0.45 – 3.45 0.68 “low” serum albumin 4.25 1.66-10.88 0.003 “high” beta 2 microglobulin 2.94 1.13-7.68 0.03 Disclosures Last: The Binding Site: Employment.