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Indtroduction and methods: In chronic lymphocytic leukemia (CLL), molecular and cytogenetic diagnostics are crucial for the determination of accurate prognosis and treatment choice. Among different genetic aberrations, del(17p13) or TP53 mutations constitute high-risk factors, and early identification of such defects is a high priority for CLL patients. While cytogenetic diagnostics is well-established and accessible for the majority of CLL patients in Poland, molecular diagnostics of TP53 mutations is performed only in a few ERIC-certified centers (eight as of September 2020), and only two of these employ next-generation sequencing (NGS) for routine analysis of TP53 status in CLL patients. Here we report the interim results of a project assessing TP53 mutations with NGS technology in relapsed or refractory CLL patients with confirmed negative del(17p13) status. 249 patients from 32 clinical centers were included in the study. Results: NGS analysis revealed TP53 mutations in 42/249 (17%) patients, half of whom (21/249, 8.5%) had subclonal mutations (VAF ≤10%). These results are in line with published data in relapsed/refractory CLL patients. Conclusions: The results of the project demonstrated the feasibility and accuracy of NGS testing in CLL patients despite several initial logistical and technical obstacles. Our study also proved that, with appropriate funding, CLL patients from any hematological center in Poland can have access to state-of-the-art molecular diagnostics

Introductionκ and λ serum free light chains (sFLCs) are produced during physiological lymphopoesis by plasmocytes and B lymphocytes in a constant ratio related to heavy chains. The measurement of sFLC plays an important role in the diagnosis and monitoring of patients with multiple myeloma (MM). The first reports suggested that sFLC disturbances might have prognostic value also in patients with chronic lymphocytic leukemia (CLL). Aim of the study: The aim of the study was to evaluate the relationship between sFLC concentration and recognized prognostic factors and clinical course of CLL. Materials and methods: The sFLC concentration was measured using a latex-enhanced immunoassay in 59 patients with newly diagnosed CLL. The relationship between sFLC concentration and time to start of the treatment (TFT), the response rate to therapy (ORR) and overall survival (OS) was assessed.ResultsA significant correlation was found between sFLC κ concentration and the clinical stage of leukemia according to Rai classification, β-2 microglobulin concentration, LDH activity, CD38 expression, as well as between sFLC λlevel and β-2 microglobulin concentration and platelet count (PLT ). There was also a correlation between the values of summated κ and λ and the clinical stage of disease according to Rai classification, β-2 microglobulin concentration, CD38 expression, white blood cells count (WBC), lymphocyte count (ALC) and hemoglobin (Hgb) concentration. The κ/λ ratio (FCLR) values were significantly different in the CD38+ and CD38- population.SummarySimple and reproducible clonality index, which constitutes the sFLC concentration assessment, can be an attractive, potential prognostic marker in patients with CLL, however further studies are needed on a larger group of patients especially in relation to the predictive value of sFLC.

StreszczenieZespół hemofagocytowy (ang. hemophagocytic lymphohistiocytosis – HLH) charakteryzuje się nieprawidłową aktywacją układu immunologicznego, u podłoża której leżą zmiany genetyczne lub nabyte zaburzenia cytotoksyczności limfocytów T i NK. Objawy kliniczne są niespecyficzne i różnorodne, a postawienie rozpoznania, pomimo dostępności badań dodatkowych, jest niezwykle trudne. W artykule zaprezentowano przypadek kliniczny chorej, u której pierwotnie w obrazie klinicznym dominowała nawracająca gorączka oraz zmiany skórne sugerujące rozpoznanie rumienia guzowatego. Pomimo rozpoczęcia steroidoterapii oraz stosowania empirycznej antybiotykoterapii stan pacjentki nie ulegał poprawie. Do objawów klinicznych dołączyły się splenomegalia oraz zaburzenia w badaniach dodatkowych: trójukładowa cytopenia, hiperferrytynemia, hipertriglicerydemia, hipofibrynogenemia. Ponadto w obrazie histopatologicznym szpiku kostnego stwierdzono obecność hemofagocytów. Na podstawie obrazu klinicznego oraz badań dodatkowych postawiono rozpoznanie HLH. W terapii zastosowano chemioterapię zgodnie z protokołem HLH-2004, uzyskując całkowitą remisję.

Introduction In peripheral blood, monocytes form a heterogeneous population of cells. One particular subset of circulating monocytes is expressing the angiopoietin receptor Tie-2 (Tie2-expressing monocyte; TEM). TEM are characterized by tumor promoting properties. However, the role of TEM in chronic lymphocytic leukemia (CLL) immunopathogenesis remains undefined. Material and Methods Here, we evaluated the monocytes with Tie-2 expression (CD14 + Tie-2 + ) in peripheral blood of CLL patients (n=55) and normal subjects (n=15) by flow cytometry. We investigated possible associations between TEM and poor prognostic factors such as CD38 or ZAP-70 expression, Rai stage and unfavorable cytogenetic abnormalities. Moreover, we investigated the association of TEM percentage with CD14 ++ CD16 + monocytes and Treg percentages. Results We found that CLL patients had a higher percentage of CD14 + Tie-2 + monocytes compared to normal controls. The percentage of TEM was positively associated with ZAP-70 expression as well as with unfavourable cytogenetic changes: del(17p) and/or del(11q). The frequency of TEM increased with the disease stage. We showed no correlation between the percentage of TEM and CD38 expression. The percentage of TEM at diagnosis was associated with white blood cell count as well as with the percentages of CD19 + CD5 + lymphocytes and Tregs. The majority of CD14 + Tie-2 + cells belonged to the intermediate monocytes subset (CD14 ++ CD16 + ) while fewer of them were among the classical (CD14 ++ CD16 − ) or non-classical monocyte (CD14 + CD16 ++ ) subsets. TEM and CD14 ++ CD16 + monocytes have a proangiogenic activity, suppress T-cell activation and promote Treg expansion. The results suggest that monitoring of TEM number and function may provide useful information in determining disease activity.

Chronic lymphocytic leukemia (CLL) which is the most common leukemia in adults in Western countries still remains incurable. There is an intense search for the prognostic markers that might facilitate the treatment of patients according to individual prognosis. The aim of the presented study was to evaluate the expression of CD1d molecule on peripheral blood B cells from 70 untreated patients with CLL and 20 healthy donors. The samples were analyzed by flow cytometry directly after preparation. The results of the study showed that the median percentage of CD1d-positive B cells was significantly lower in peripheral blood of patients with CLL than in healthy subjects from control group. Additionally, the percentage of CD1d+ B cells in CLL patients varied in patients with different Rai stages. We have also observed significant differences in CD1d expression depending on CD38 or ZAP70 expression. Moreover, patients with CLL had lower percentages of iNKT cells than healthy donors and the percentage of CD1d+/CD19+ cells inversely correlated with the percentage of iNKT cells. Our results suggest the important role of CD1d molecule in the development and progression of CLL, as well as its potential prognostic significance.

Functional disorders of T lymphocytes play an essential role in abnormal immune response in patients with chronic lymphocytic leukemia. The aim of this study was to assess the profile of cytokines expressed by T cells derived from patients with CLL. We have demonstrated that the intracellular levels of IL-2, IL-4, IFN-γ, TNF, IL-6 and IL-10 were significantly higher in T cells of CLL patients than in healthy donors. Moreover, the percentages of CD4+/CD3+/TNF+, CD4+/CD3+/IFN-γ+, and CD4+/CD3+/IL-2+ cells were significantly higher in ZAP-70-positive patients compared with ZAP-70-negative ones. Likewise, significantly higher percentages of CD4+/CD3+/TNF+, CD4+/CD3+/IFN-γ+ cells were observed in CD38-positive than in CD38-negative cases. What is more, there was a significant difference in median percentage of CD3+/CD4+ cells expressing TNF, IL-4, IFN-γ, IL-2 or IL-6 between patients carrying the 11q22.3 deletion and/or the 17p13.1 deletion and patients without these genetic aberrations. Our results confirm the functional disorders of T cells in CLL and their influence on the clinical course of the disease.

Summary In this study we analyzed selected parameters of apoptosis in leukemic cells from peripheral blood and bone marrow of patients with chronic lymphocytic leukemia (CLL). The percentage of apoptotic leukemic B cells (Δψm low /CD19 + ) was significantly lower in peripheral blood (median: 0.99%) than in bone marrow (median: 1.41%) (p low /CD19 + cells was significantly higher in ZAP-70-positive patients than from ZAP-70-negative patients (p

M. Grzywnowicz *, L. Karabon , A. Piechnik , M. Zając , K. Skorka , J. Zaleska , W. Tomczak , P. Wlasiuk , A. Bojarska-Junak , A. Dmoszynska , I. Frydecka , K. Giannopoulos 1,3 Samodzielna Pracownia Hematoonkologii Doświadczalnej UM, Lublin, Polska Zaklad Terapii Doświadczalnej, Instytut Immunologii i Terapii Doświadczalnej PAN, Wroclaw, Polska Klinika Hematoonkologii i Transplantacji Szpiku UM, Lublin, Polska Katedra i Zaklad Immunologii Klinicznej UM, Lublin, Polska *Autor prezentujący i do korespondencji. Adres email: mgrzywnowicz@wp.pl