Background: Gal-3 is the only known member of the group of so-called chimeric galectins. It has 3 structural domains, a NH2 terminal domain with serine phosphatase activity, a repetitive collagen-like domain and a COOH terminal carbohydrate-recognizing domain. Gal-3 is thought to be involved in crucial cellular processes, including regulation of cell proliferation, apoptosis and angiogenesis. Gal-3 promotor contains NF-κB binding sites (Kadrofske et al, 1998). Gal-3 shares the BH1 domain with BCL2 and has antiapoptotic activitiy, but seems to affect different intracellular structures than BCL2 (Akahani et al, 1997). Normal memory B-cell, but not germinal-center cells, express gal-3. More than 50% of DLBCLs express gal-3, but none of Burkitt, follicular, marginal zone and small lymphocytic lymphomas (Hoyer et al, 2004). While preliminary data suggest that gal-3 + DLBCLs have inferior prognosis, its relation to other immunohistochemical markers, cell of origin (COO) and clinical characteristics of lymphomas are unknown Aims: To compare biological and clinical characteristics of gal-3 + and gal-3 – DLBCLs and thus gain insight into its biological function and clinical importance in this tumor type. Methods: We analyzed clinical characteristics (age, sex, IPI factors, response to treatment, event-free survival (EFS) and overall survival (OS)), immunohistochemical expression of gal-3, BCL2, VEGF and NF-κB (p65), and cell of origin (COO) as determined by Hans’ algorithm, in newly diagnosed DLBCL patients treated with R-CHOP or similar regimens. All markers were evaluated using commercially available monoclonal antibodies. Results: 123 patients were included in the study with a median follow-up of survivors of 68 months. Gal-3 was positive in 69 (56.1%), negative in 51(41.4%) and undetermined in 3. Patients with higher IPI, non-GC subtype and NF-κB + expressed gal-3 more frequently (Table). In univariate analysis gal-3 expression was not a statistically significant prognostic factor for OS and EFS in the whole group of patients (p=0.16 for EFS and p=0.19 for OS). However, BCL2 + patients fared significantly worse if they coexpressed gal-3 (EFS at 2 y 44% vs 73% (p=0,002) OS at 2 y 50% vs 74% (p=0,006)). Gal-3 expression had no influence on the outcome of BCL2- patients. In multivariate analysis of influence of immunohistochemical markers on EFS and OS, BCL2 was the only significant factor, while gal-3 showed a strong trend (p=0.068 for EFS and 0.093 for OS). If IPI was introduced into the model, gal-3 expression lost its significance. Image:Summary/Conclusion: Our results are in accordance with experimental data indicating that NF-κB stimulates gal-3 expression and that gal-3 potentiates the antiapoptotic effect of BCL2. Gal-3 is more frequently expressed in advanced-stage DLBCLs and those of non-GC subtype, consistent with its putative proliferative/antiapoptotic effect and its expression in memory B- but not germinal-center cells.

Background: Patients with hematologic malignancies have a high risk of dying from COVID19 due to inability to mount humoral and cellular immune responses to the virus. Remdesvir is an inhibitor of viral RNA-polimerase. Some, but not all studies suggest it hastens recovery and reduces mortality in patients with COVID19. In a large randomized trial, convalescent plasma obtained from persons recovering from the infection was not proven to be useful in treatment of COVID19 in the general population, but other studies suggest it is useful in hematologic patients unable to produce antibodies against the virus. Recommendations on the use of these two drugs vary, some recommend its use only in severely immunocompromized individuals, some only in serious cases and some not at all. Reflecting these differences, the practice of using them varied between Croatian centers during the current pandemic. Aims: To analyze the effect of remdesvir and convalescent plasma on mortality in patients with hematologic malignancies by performing a matched-pair analysis. Methods: KroHem, the Croatian Cooperative Group for Hematologic Diseases, collected data on the outcome of patients with hematologic malignancies who became infected with SARS-COV2 while on concurrent systemic antineoplastic therapy during 2020 and 2021, before the appearance of the omicron strain. Patients treated with remdesvir and/or convalescent plasma were matched to those untreated according to age, disease type and antineoplastic therapy, factors found in our previous analyses to be related to outcome. Patients with Hodgkin lymphoma and myeloproliferative neoplasms were excluded, due to low risk of COVID19 mortality. Death during infection was considered as due to COVID19. Results: We identified 119 patients fulfilling the entry criteria. Three could not be matched, 2 with T-PLL treated with alemtuzumab and one with plasmablastic lymphoma and newly diagnosed HIV infection. All three died. In the remaining 116 patient pairs remdesvir significantly reduced the mortality: 36 out of 106 treated patients died, in comparison to 54 untreated (p=0.0207, McNemar’s test). The effect of plasma was not significant: 26 of 73 treated patients died, in comparison to 33 untreated (p=0.2812). Therapy was substantially more effective in patients who received treatment within a week from symptom onset; 11 of 58 patients treated with remdesvir died in comparison to 33 untreated (p<0.0001) and 8 out of 35 treated with plasma in comparison to 20 untreated (p=0.0095). Patients treated with remdesvir only had similar outcomes as those treated with remdesvir and plasma (15% vs. 19% respectively). Image:Summary/Conclusion: Our study suggests that patients with hematologic neoplasia, who are at a high risk of dying from COVID19, should receive treatment with remdesvir and convalescent plasma as soon as possible, resulting in a 2.5-3 times reduction in mortality. The effect of later treatment, if any, is less prominent.

Background: Background: Central nervous system (CNS) relapse in multiple myeloma (MM) is a rare complication with dismal prognosis. It primarily affects transplant-eligible patients, and the incidence seems to be higher in the era of novel antimyeloma drugs. Aims: Aims: We aimed to assess clinical characteristics, treatment choices, and survival of these patients. Methods: Methods: This is a retrospective study by the Croatian Cooperative Group for Hematological malignancies (KROHEM). Patients with MM treated with novel agents prior to CNS relapse were included. Results: Results: From April 2018 to October 2021, 13 patients from eight centers were identified. There were 8 men and 5 women, with a median age of 61 years (range 47-70 years). A median time from diagnosis to CNS progression was 3 years (ranging from 4 months to 7.5 years). Patients were treated with 1 to 5 lines (median 3) of systemic therapy prior to CNS progression. The patients progressed during treatment or maintenance with bortezomib-based regimen (3 pts), carfilzomib (2 pts), lenalidomide (3 pts), daratumumab (2 pts), pomalidomide (1 pt), bendamustine (1 pt), or in treatment-free remission (1 pt). Six patients had >10% plasma cells in bone marrow, whereas only a single patient had plasma cell leukemia. Nine patients had plasma cells in cerebrospinal fluid (CSF) and were treated with intrathecal therapy – median time to CSF clearance was two to three weeks. Nine patients had osteodural myeloma mass and 7 were irradiated. Systemic treatment was administered in 12 patients with CNS penetrating agents such as bendamustine + IMiDs, daratumumab +/- IMiDs, or other chemotherapy such as DT-PACE. At the time of analysis, three patients are alive with survival 3.5, 4.5, and 8 months from CNS relapse, respectively. Two of them were autografted with BEAM or melphalan/thiotepa conditioning. The median overall survival for all patients was 3 months (1-38 months). The longest survival had two patients treated with systemic therapy and local irradiation who died 11 and 38 months after CNS relapse, respectively (image).

Background: Follicular lymphoma (FL) is a systemic neoplasm of the lymphoid tissue arising from B cell proliferation. The novel monoclonal anti-CD20 antibody obinutuzumab in combination with chemotherapy has been widely accepted as the first choice in front line treatment of FL. Severe acute respiratory syndrome coronavirus 2 (SARS‐CoV‐2), responsible for coronavirus disease 2019 (COVID-19) is causing increased mortality among patients with lymphoproliferative disorders compared with the general population. Furthermore, there are some concerns in terms of morbidity and mortality for patients with FL because of their immunocompromised status induced by recent exposure to cytotoxic chemotherapy, especially bendamustine and anti-CD20. Aims: To investigate efficacy and safety of immunochemotherapy protocols for patients with newly diagnosed FL during COVID-19 pandemic. Methods: We retrospectively investigated medical data of all patients with newly diagnosed FL grade 1, 2 or 3A from Croatian hematologic registry in period from April 2019 to March 2021. Only patients which required systemic treatment were included in the analysis. All patients received obinutuzumab (G) in combination with either CHOP, bendamustine (B) or CVP chemotherapy protocol. Treatment response was evaluated using international lymphoma response criteria. Results: We analyzed a total of 114 FL patients treated with G-chemotherapy. Mean age was 62.4 ±10.5 years. Majority of patients were female (71/114 (62.3%)). FL grade I was present in 45/114 (39.5%), grade II in 28/114 (24.6%), grade III in 27/114 (23.7%) and not specified (but not IIIB) in 14/114 (12.3%) patients. A total of 61/114 (53.5%) patients were treated with G-B, 49/114 (43%) with G-CHOP and 4/114 (3.5%) with G-CVP immunochemotherapy. Similar rates of adverse events were observed in patients treated with G-CHOP and G-B Median follow up was 17 months. Overall response rate was 94%, complete remission (CR) in 68% and partial remission (PR) in 25% of patients. Median overall survival (OS) and progression free survival (PFS) were not reached with 12-months rates of 94% and 92%, respectively. Patients treated with G-CHOP had statistically significantly superior OS and PFS compared to patients treated with G-B (P=0.002 and P=0.006, respectively, Fig. 1). More favorable survival course associated with G-CHOP in comparison to G-B persisted in multivariate analysis (P=0,026, HR=15,12) after adjustment for age, sex, FLIPI grade and SARS-CoV-2 infection. Total of 12 patients died during the follow up and COVID-19 was cause of death in 5 patients. During the follow-up SARS-CoV-2 infection was diagnosed in 20/114 (17,5%) patients with overall mortality rate of 25%. All of the 7 patients treated with G-CHOP recovered from SARS-CoV-2 infection and mortality rate in infected group of patients treated with G-B was 33% (4/12 patients). Image:Summary/Conclusion: Increased COVID-19 mortality in patients with lymphoproliferative disorders was observed in this study. Our group of patients had reduced OS and PFS compared to the GALLIUM trial and SARS‐CoV‐2 infection was the most pronounced risk factor for death. Even though in some studies bendamustine has shown to be less toxic and more effective than CHOP in FL, there are some important pandemic aspects that must be considered. Bendamustine exposure seems to be associated with worse outcome in case of the infection with SARS-CoV-2. These intriguing differences could play important role in treatment approach in COVID-19 pandemic. Future studies investigating hematological malignancies in COVID-19 pandemic are warranted.

Background:Autoimmune hemolytic anemia (AIHA) is a rare autoimmune disease in which red blood cells (RBC) are targeted by autoantibodies confirmed with positive direct antiglobulin test (DAT), resulting in premature destruction of RBC. AIHA may be subdivided into warm antibody AIHA (wAIHA) and cold antibody AIHA (cAIHA), and also to primary and secondary depending on the presence of an underlying disorder. In addition to that, patients may have positive DAT but without features of hemolysis, and some of them will later develop AIHA.Aims:The aim of this study was to analyze characteristics of newly diagnosed DAT positive AIHA patients during the 5 years period in our tertiary center, as well to assess DAT positive patients without features of AIHA.Methods:This was a single‐center retrospective cohort study performed at the University Hospital Center (UHC) Zagreb, Zagreb, Croatia, Department of Internal Medicine, Division of Hematology. We included data for all consecutive adult patients with a newly diagnosis of DAT positive AIHA between 2014 and 2018, and also all DAT positive patients without features of hemolysis in that time period. This study was approved by the UHC Ethical Committee. Descriptive statistics was performed and included means ± SD or median (range) as appropriate for continuous variables and frequency (percentage) for categorical variables.Results:The data from 56 patients (50% women) with newly diagnosed DAT positive AIHA during the study period were analyzed. The median age at time of AIHA diagnosis was 67 years (range 22–90 years). Majority of AIHA was wAIHA (51 (91.1%) patients). AIHA was considered primary in only 14 (25%) patients and was associated with an underlying disorder in 42 (75%) patients. The various disorders were associated with secondary wAIHA, the most frequently B‐cell lymphoproliferative malignant diseases (33%), solid tumors (9.8%), myelodisplastic syndrome (9.8%) and autoimmune diseases (9.8%). Median hemoglobin level at the AIHA diagnosis was 71 g/L. 82.1% of patients received RBC transfusion. Seven (12.5%) patients did not require immunosuppressive treatment and other received mostly corticosteroids as the 1st line of therapy. Rituximab received 9 patients (17.6%) with wAIHA, mostly as the 2nd line of treatment. Eight patients (14.3%) with AIHA developed thrombosis (7 with wAIHA and 1 with cAIHA). During the 5‐years study period additional 120 patients with DAT positive results but without signs of hemolysis were detected.Summary/Conclusion:Data from our 5‐years single centre experience with AIHA are comparable with literature data. Majority of patients had secondary AIHA and wAIHA. Rituximab is used mostly as the second‐line treatment and corticosteroid‐sparing agent for wAIHA. Significant number of patients with DAT positive results but without features of hemolysis was detected, and they need to be closely monitored for possible development of AIHA.

Background:Primary and secondary myelofibrosis (PMF and SMF) are clonal hematopoietic stem cell disorders characterized by myeloproliferation, bone marrow fibrosis, splenomegaly and a strong inflammatory milieu. Glycoprotein YKL–40 emerged as an important biomarker of inflammation, angiogenesis, tissue remodeling, cell proliferation and differentiation. Serum YKL‐40 levels were shown to be higher in PMF patients when compared to healthy controls. However, the clinical significance of this finding is unknown.Aims:The aim of our study was to investigate serum YKL‐40 levels in PMF and SMF patients and to assess its clinical correlations.Methods:Using commercially available ELISA kit (R&D Systems Europe, Abingdon, UK) we analyzed serum YKL‐40 levels in 30 myelofibrosis patients (13 PMF and 17 SMF) and in 25 sex‐ and age–matched healthy controls. Correlations with clinical parameters were made. Optimal cut ‐ off for survival analyses was determined using the ROC curve analysis. The Kruskal‐Wallis analysis of variance, the Mann‐Whitney U test, the Spearman rank correlation, the log rank test and the Cox regression analysis was used. P values <0.050 were considered statistically significant for all analyses.Results:Serum YKL‐40 levels significantly differed between patients and controls (p = 0.006): both PMF (median 685.3 pg/mL, range 220 – 4000) and SMF (median 1797.8 pg/mL, range 58 – 4000) patients had higher serum YKL‐40 levels in comparison to controls (median 560 pg/mL, range 117 – 1137; p<0.050 for both comparisons), whereas there was no statistically significant difference between PMF and SMF patients. Higher serum YKL‐40 levels were statistically significantly associated with presence of constitutional symptoms (median 465.1 pg/mL vs 3694.5 pg/mL for patients with or without constitutional symptoms; p = 0.001), higher Eastern Cooperative Oncology Group (ECOG) performance status (median 685.3 pg/mL vs 3413.2 pg/mL for patients with 0‐1 and 2‐4 performance status, respectively; p = 0.047), higher platelet count (rho 0.390; p = 0.033), larger spleen (rho 0.750; p<0.001), lower serum lactate dehydrogenase (LDH) (rho ‐0.380; p = 0.039) and less prominent bone marrow fibrosis grade (rho ‐0.480; p = 0.007). Patients receiving ruxolitinib had lower serum YKL‐40 levels (median 552.9 pg/mL) than those on hydroxycarbamide therapy (median 4000 pg/mL; p = 0.005). In univariate survival analysis, patients with higher serum YKL‐40 levels (>2311.15 pg/mL) had inferior overall survival in comparison to patients presenting with lower YKL‐40 levels (≤2311.15 pg/mL) (HR 2.91; p = 0.034). This association remained significant in the multivariate Cox regression model after adjusting for sex, age, DIPSS score and ruxolitinib therapy (HR 5.59; p = 0.029).Summary/Conclusion:This is the first study to report clinical associations and strong prognostic properties of increased serum YKL‐40 levels in patients with parimary and secondary myelofibrosis. Serum YKL‐40 levels might represent a state of pronounced inflammation and increased tumor burden in myelofibrosis, as they are significantly associated with presence of constitutional symptoms, poor performance status and parameters indicative of stronger myeloproliferation. Additional studies are needed to more precisely elucidate the cell(s) of YKL–40 origin and to clarify the role of YKL‐40 in promoting disease progression and bone marrow fibrosis. Prognostic properties of YKL‐40 also need to be confirmed in larger prospective cohorts of uniformly treated myelofibrosis patients. Nevertheless, our results identify YKL‐40 as a potential prognostic biomarker in myelofibrosis.

Background:Thromboembolism frequently complicates treatment of acute lymphoblastic leukemia (ALL).Aims:A single‐center retrospective study was conducted with the objective of assessing the risk factors for thromboembolism in adult patients with ALL.Methods:The study included 87 consecutive patients (aged 19–70 years; 57 males) treated for ALL; B‐cell (n = 40), T‐cell (n = 26), Ph+ (n = 13), byphenotypic (n = 3) and unclassified (n = 5) during the 10‐year period. All patients but one received intensive treatment, including L‐asparginase in 57 (65%) patients, while 23 (26%) underwent allogenic stem cell transplantation. Vast majority of patients (n = 80, 92%) had central venous catheters at least once during the treatment period, including peripherally inserted central catheter (PICC) placed in 18 (21%) patients.Results:Fourteen patients (16%) developed thrombosis during the study period. Deep venous thrombosis was described in 9(65%), pulmonary embolism in 2(14%), and cerebral sinus thrombosis in 3(21%) patients. One death was attributable to thromboembolism. The risk of thromboembolism was associated with the induction treatment, protocols including L‐asparginase, the use of central venous catheters, namely PICC, and non‐0 blood groups.Summary/Conclusion:Our results demonstrate high incidence of thromboembolic events in patients with ALL. The highest risk is attributed with the use of L‐asparginase, despite vigorous control of coagulation parameters and antithrombin substitution. Finally, our results strongly suggest against simultaneous use of L‐asparginase and PICC.

Background:Cardiovascular and thromboembolic events are the leading causes of morbidity and mortality for patients with myeloproliferative disorders (MPN).Aims:This retrospective study aimed to evaluate the incidence and the risk factors for the arterial and venous thrombosis in treated patients with MPN.Methods:The study included 329 adult MPN patients, 184 (56%) female, treated at University Hospital Center Zagreb, Department of Internal medicine, Division of Hematology and Zadar General Hospital, Hematology Unit. Patients were aged 24‐85 years (median 68), and were diagnosed as having essential thrombocythemia (ET) (n = 149, 45%), polycythaemia vera (PV)(n = 94, 29%), primary myelofibrosis (PMF)(n = 69, 21%) and unclassifiable MPN (n = 17, 5%). Diagnosis were made according to the 2008 WHO Classification. JAK‐2 mutation status was either established or revised in our center, but was unknown in 55 (17%) patients. Data about treatment and cardiovascular and thromboembolic incidents was retrieved from medical records.Results:Forty patients (12%) developed thrombosis after median of 36 (range 1‐240) months from the time of diagnosis. Fifteen (5%) patients developed venous thrombosis, twenty‐four (7%) arterial, while one (0,3%) patient both type of thrombosis. Regarding the disease type, thrombosis was described in 16 patients with ET (11%), 18 with PV (19%) and 6 with PMF (8%). The risk of thrombosis was the highest in patients with previous thrombotic events, but did not correlate with the type of therapy (cytoreductive and/or antiagreggation) nor the JAK‐2 status.Summary/Conclusion:The risk of both arterial and venous thrombosis was found to be increased in patients with MPN even after introducing appropriate treatment. The highest risk of post treatment thrombosis was found in patients with previously diagnosed thrombotic events.

A retrospective analysis was performed in patients stratified according to GCSF used for mobilisation (Neupogen vs Nivestim) and the hematological malignancy they were treated for (Acute myeloid leukemia, Multiple myeloma, non- Hodgkin`s lymphoma, Hodgkin`s lymphoma) patients who were administered 10 mcg/kg of GCSF were analysed. The following parameters of stem cell mobilisation were surveyed: number of days GCSF was administered, total number of CD34 positive cells per kilogram collected, total number of colony forming units of the granulocyte macrophage order per kilogram collected. A matched pairs analysis was performed on a subset of patients treated for multiple myeloma.

BACKGROUND: Therapy of multiple myeloma (MM) has been greatly advanced by introduction of autologous hematopoietic stem cell transplantation (AHSCT) in 90’s, and appearance of new potent drugs thalidomide, bortezomib and lenalidomide in the last decade. AIMS: to assess the efficacy of new treatment modalities on survival of MM patients treated at the University Hospital Center, Zagreb. PATIENTS AND METHODS: From 1985 till 2010, 560 consecutive MM patients (pts) were analyzed. Median age at diagnosis was 60 (range 28-89) years. Pts were divided into three groups according to time period of available treatment modalities: Group one treated from 1985-1995, period prior AHSCT (n=158, median age 63 [range: 31-87]) ; group two treated from 1996-2001, period with AHSCT (n=139, median age 58 [range: 29-87]) ; and group three treated from 2002-2010, period with AHSCT+bortezomib+thalidomide (n=263, median age 60 [range: 28-89]). Each group was subdivided in two subgroups according to age at diagnosis indicating eligibility for AHSCT (<65 and ≥65 years of age). Estimated median overall survival (OS) was calculated by Kaplan-Meier method. Differences between groups were tested by 2-tailed log-rank test with p value of <.05 being statistically significant. RESULTS: Median follow-up for entire cohort was 42.5 (range: 1-267 months). Seventy-three pts were lost from follow-up after median time of 16 (range: 1-123) months. Estimated median OS was 69 (95%CI 59.07-78.93) months. For groups 1 and 2, estimated median OS were 38 (95%CI 29.44-46.56) and 47 (95%CI 36.06-57.94) months respectively, while for the group 3 estimated median OS was not reached for the median follow-up of 48 (range: 1-118) months. OS was significantly different between all groups: groups 1:2 (p=0.009), groups 2:3 (p<0.0001), groups 1:3 (p<0.0001) (Fig. 1). When adjusted for age, significance between groups 1 and 2 was not reached (p=0.058) although showing trends for better OS in group 2. Statistically significant difference remained between groups 1:3 and 2:3 (p<0.0001, for each). OS for patients younger than 65 are significantly better compared to patients with 65 years or older, in all three groups. CONCLUSIONS: The analysis of OS clearly showed significantly better outcome for pts treated with AHSCT, especially for younger patients. In most recent time period both younger and older patients showed better OS probably due to introduction of new treatment modalities with thalidomide or bortezomib. Results of the study are in accordance with other similar retrospective analyses.

THE VALUE OF NEGATIVE F-18-FDG-PET IN PATIENTS WITH HODGKIN'S DISEASE AND A RESIDUAL MASS AFTER THERAPY Dražen Huic, Andrea Mutvar, Sandra Kinda-Basic, Igor Aurer, Martina Ciglar, Darko Grosev, Ivo Radman, Boris Labar, Damir Dodig Department of Nuclear Medicine and Radiation Protection, Department of Internal Medicine, Division of Hematology, Zagreb University Hospital Centre, Zagreb, Croatia Aim: To asses the negative predictive value (NPV) of FDG-PET performed with triple-head coincidence gamma camera after first-line therapy or salvage therapy in patients with Hodgkin’s disease (HD) compared by long-term follow-up as a reference standard. Design and Setting: This single centre retrospective diagnostic test study was done in University Hospital Centre between June 2001 and February 2008. Participants: The charts of 131 consecutive patients with Hodgkin’s disease were reviewed. Seventy-three consecutive PET-negative patients (median age 28 years ; range 12-80 years) with primary or recurrent biopsy confirmed lymphoma after first-line therapy or salvage therapy were followed-up at least 12 months (median 23 months ; range 12-69 months). All already performed 18F-FDG PET scans (using hybrid PET camera with triple head coincidence imaging capability within a few months after completion of therapy) were again visually interpreted by two board-certified nuclear medicine physicians who were blinded to any clinical or CT data. Main outcome measure: The negative predictive value of FDG-PET performed with triple-head coincidence gamma camera (Index test) was compared with long-term follow-up as a reference standard. Results: Out of 131 patients 73 turned-out to be PET-negative. Of those 73 PET-negative patients 61 patients have been scanned after first-line chemotherapy/radiotherapy, and only 3 (4.9%) of them relapsed in a follow-up (negative predictive value 95%). Twelve patients with resistant disease have been scanned after repeated therapy, and 4 (25%) of them relapsed in a follow-up period (negative predictive value 66%). Conclusions: This methodology with triple-head coincidence gamma camera has high negative predictive value in patients with Hodgkin's disease.