Background We investigated serous effusions occurring during the course of an already known hematologic neoplasia or as a first manifestation of it. We correlated cytology results with flow cytometry results, when available. In the absence of flow cytometry, our correlation was based on clinical follow up information obtained retrospectively. We evaluated our results in relation to the data of the literature and we considered some new suggestions for the improvement of cytology service. Methods Serous effusions in hematologic patients were retrieved from the files of the Department of Cytology, Laiko Hospital, for a period of 2 years. All patients had enrolled either a previous hematologic history, or a suspicious clinical and imaging status. Seventy‐three serous effusions were included. Cytology reports consisting of morphology and immunocytochemistry assessment were correlated to flow cytometry results and, occasionally, to clinical follow‐up. Results In the group of patients with previous history, sensitivity was 82.76%, positive predictive value was 100%, specificity 100%, and negative predictive value was 58.33%. In the group of patients without previous history, sensitivity and positive predictive value were both 91%, whereas specificity and negative predictive value could not be estimated. Conclusion We provide evidence that the diagnostic accuracy of cytology with the adjunct of immunocytochemistry is high compared to flow cytometry for detecting hematologic malignancies. In order to improve clinical performance, it is suggested that a cytology triage of serous effusions in all patients with hematologic malignancy must be implemented.

CD69 is the earliest activation marker newly synthesized and expressed during T lymphocyte activation. In this study, a whole-blood flow-cytometry-based assay was used to assess expression of the activation antigen CD69 on CD4 and CD8 T lymphocytes, and the co-expression of CD69 and CD28 on T cells. The expression of CD69 was studied in both unstimulated and in phytohaemagglutinin (PHA)- or anti-CD(3)/CD(28)-stimulated, 4-h culture, samples. The production of IL-2, IFN-gamma or both cytokines, in CD69(+) T cells, in response to Staphylococcus enterotoxin B was also tested. Fifty-three HIV-1-infected and 21 healthy volunteers participated in this study. In both PHA- and anti-CD(3)/CD(28)-stimulated cultures the percentage of CD69 on CD3(+)CD4(+) T cells was significantly lower in AIDS (and non-responders to HAART) versus healthy controls and the other HIV-1(+) groups. A decrease of CD69(+)CD28(+) T cells after PHA or MoAbs stimulation is noticed in AIDS. No difference in cytokine production was noticed between healthy volunteers and HIV-1(+) patients. Our results suggest that the expression of CD69 is affected only in the AIDS stage and in the non-responders to HAART patients.

Myelodysplastic syndromes (MDS) encompass a very heterogeneous group of clonal hematopoietic stem cell differentiation disorders with malignant potential and an elusive pathobiology. Given the central role of metabolism in effective differentiation, we performed an untargeted metabolomic analysis of differentiating myeloid lineage cells from MDS bone marrow aspirates that exhibited <5% (G1) or ≥5% (G2) blasts, in order to delineate its role in MDS severity and malignant potential. Bone marrow aspirates were collected from 14 previously untreated MDS patients (G1, n = 10 and G2, n = 4) and age matched controls (n = 5). Following myeloid lineage cell isolation, untargeted mass spectrometry-based metabolomics analysis was performed. Data were processed and analyzed using Metabokit. Enrichment analysis was performed using Metaboanalyst v4 employing pathway-associated metabolite sets. We established a bioenergetic profile coordinated by the Warburg phenomenon in both groups, but with a massively different outcome that mainly depended upon its group mitochondrial function and redox state. G1 cells are overwhelmed by glycolytic intermediate accumulation due to failing mitochondria, while the functional electron transport chain and improved redox in G2 compensate for Warburg disruption. Both metabolomes reveal the production and abundance of epigenetic modifiers. G1 and G2 metabolomes differ and eventually determine the MDS clinical phenotype, as well as the potential for malignant transformation.

Dissemination of lymphomas in serous effusions is quite common. Cytology aims to contribute in the clinical management of haematologic patients, providing an accurate and rapid diagnosis. Ancillary techniques such as immunocytochemistry and flow cytometry are essential to classify the lymphoma entity. Comprehensive awareness of the clinical picture and previous histologic documentation are essential for a lymphomatous effusion diagnosis. We report an unusual case of monomorphic epitheliotropic intestinal T-cell lymphoma, formerly known as enteropathy associated T-cell lymphoma (EATL) type II, spreading in the pleural cavity. Cell morphology and immunohistochemistry of the pleural fluid were consistent with the histology of a jejunal tumor previously excised. Flow cytometry data were consistent, though not pathognomonic for the disease. Serous effusions with evidence of lymphoma involvement should be thoroughly examined with cytology and adjuvant techniques to provide diagnosis for proper therapeutic strategies.

ATLL is etiologically associated with HTLV-I retrovirus. A population of 10 to 20 million worldwide is estimated to be infected by the virus, but only 1-4% develop ATLL during a 70-year lifespan. The latency period is more than 30 years. The aim of this study was to report two cases of ATLL in Greek patients with the concomitant study of their family members. A 55-year-old woman and a 59-year-old man presented with leucocytosis and lymphocytosis. Both were asymptomatic and physical examination was unremarkable except for minimal lymphadenopathy in the second patient. In both patients blood smears showed small-to-medium-sized, multilobulated lymphocytes, with different degrees of nuclear irregularity. Immunophenotypic study was as follows: CD2 + (97%), CD3 + (95%), CD5 + (95%), CD3/CD4 + (93%), CD3/CD25 + (84%), CD7 -/CD4 + (89%) CD2 + /HLA-DR + (53%), TCRabeta + (96%) and CD7-(7%). Bone marrow biopsy revealed a normal cellularity with dyserythropoiesis and scattered small lymphocytes (CD4 + on immunostaining) Serum HTLV I and II antibodies were positive. T-cell receptor gamma-chain rearrangement was positive in blood lymphocytes by PCR. Cytogenetic analysis showed complex karyotypic abnormalities. DNA analysis by PCR demonstrated the integration of the HTLV-I DNA in the DNA of the neoplastic T cells. Both patients rapidly developed acute type ATLL. In the first patient multiple subcutaneous nodules on the palmar surface of both hands were also observed. She received deoxycoformycin, which was stopped because of autoimmune hemolytic anemia. Corticosteroid treatment was initiated, with gradual improvement. She suffered from recurrent opportunistic infections. She is currently under interferon and zidovudine therapy with stable blood parameters. Chemotherapy was administered to the other patient with > 50% initial response. Both patients' families were tested for serum anti HTLV-I antibodies and their mates were found to be positive; they also had detectable viral DNA by PCR analysis while asymptomatic, with no abnormal clinical findings and normal white blood cell count and morphology. In conclusion, the two aforementioned patients are the first fully documented ATLL patients described in Greece. Investigation for HTLV-I antibodies should be mandatory in all patients with T-cell lymphoproliferative disorders.

Signal transducer and activator of transcription (STAT) proteins have been intensively studied in hematologic malignancies, and the efficacy of agents against STATs in lymphomas is already under research. We investigated the expression of total STAT5 and STAT5b in peripheral blood samples of patients with chronic lymphocytic leukemia (CLL) in correlation with the presence of Epstein–Barr Virus (EBV) and its major oncoprotein (latent membrane protein 1, LMP1). The EBV load was measured in the peripheral blood by real‐time PCR for the BXLF1 gene and the levels of LMP1 by PCR and ELISA. Western blotting was performed for total STAT5 and STAT5b in protein extracts. STAT5b was only expressed in patients (not in healthy subjects) and STAT5 but particularly STAT5b expression was correlated with the presence of the virus (77.3% vs. 51.2%, P = 0.006 for STAT5b) and to the expression of LMP1 (58.3% vs. 21.6%, P = 0.011 for STAT5b). Moreover, the expression of STAT5b and the presence of EBV and LMP1 were strongly negatively correlated with the overall survival of the patients (log‐rank test P = 0.011, 0.015, 0.006, respectively). Double positive (for EBV and STAT5b) patients had the lowest overall survival (log‐rank test P = 0.013). This is the first report of a survival disadvantage of EBV+ patients with CLL, and the first time that STAT5b expression is correlated with survival. The correlation of STAT5 expression with the presence of the virus, along with our survival correlations defines a subgroup of patients with CLL that may benefit from anti‐STAT agents.

We have analyzed the immunohistochemical expression of a wide range of molecules along with the proliferation rate separately in the proliferation centers (PCs) and in the rest of the tumor area, in lymph node or spleen sections of patients with chronic lymphocytic leukemia (CLL). Fas, FasL and c-FLIP were observed both within and outside the PCs in all cases. However, only the difference in FasL expression between the PCs and the non-PC areas attained statistical significance. Median survivin expression in the PCs was higher compared to the non-PC areas. Cleaved caspase 3 was expressed at very low levels both within and outside PCs, while BCL-2 protein was expressed at high levels in all cases in both tumor compartments. Multivariate analysis demonstrated that concurrent overexpression of Fas/FasL/c-FLIP in the PCs was correlated with worse outcome for progression-free survival as well as for overall survival.

Background: Hypoxia is a prominent feature of the BM microenvironment, influencing both normal and malignant hematopoiesis. HIF-1α, which is a key regulator of hypoxia responses by mediating the transition to glycolytic metabolism, serves as a cell cycle checkpoint of HSC quiescence and function. It has been proposed that differential HIF-1α protein expression between hypoxic endosteal and less hypoxic vascular niche finely regulates normal hematopoiesis by promoting both quiescence and survival of HSCs, as well as proliferation and differentiation of HPCs. DNA damage response 1 gene (REDD1) is a direct transcriptional target of HIF-1α linking hypoxia to energy regulation and autophagy. Recent evidence suggests that metabolism and autophagy are developmentally programmed and essential for effective hematopoiesis. Aims: To study the implication of HIF-1α/REDD1/autophagy/metabolism axis in differentiation/maturation of hematopoietic BM cells of MDS patients. Methods: BM aspiration and biopsy samples were collected from 15 untreated MDS patients from all subtypes except MDS-RARS and 7 age-matched controls with non-malignant hematologic disorder. Demographic, clinical, laboratory and karyotypic parameters were recorded. BM biopsies were immunohistochemicallly stained by fluorescent-labeled 2-nitroimidazole to assess hypoxic areas in BM. CD34 and myeloid lineage cells were isolated using magnetic beads and ficoll double-layer protocol, respectively. BM cell populations were determined by FACS analysis using standard gating strategies. HIF-1α and REDD1 gene and protein expression was evaluated by qRT-PCR and FACS analysis, respectively. Autophagy was determined by immunofluorescence for LAMP-1/LC3B and immunoblotting for LC3B/p62 (SQSTM1), whereas mitophagy by immunofluorescence for LC3B/TOMM20. Mitochondrial membrane potential (ΔΨ) and mitochondrial mass were analyzed by FACS analysis using mitotrackers. Metabolomic analysis of myeloid lineage cells was performed by liquid chromatography mass spectrometry (LC-MSn). Raw data files were processed using several chemo-informatics tools. Results: We found a preferential strong accumulation of 2-nitroimidazole in intrasinusoidal regions of MDS BM, indicating that hypoxia is a fundamental feature of BM in MDS. We demonstrated a statistically significant REDD1 gene overexpression and an increased intracellular protein co-expression of HIF-1α and REDD1 protein levels in both CD34 and myeloid cells from MDS compared to controls, as determined by RT-qPCR and FACS analysis, respectively. Higher REDD1 protein expression was shown in patients with high grade dysplasia as assessed by the Ogata classification system. Moreover, both CD34 and myeloid cells from MDS demonstrated increased LC3B puncta compared to controls with concurrent staining for CD34 and MPO. The quantitative evaluation of LC3B by Western blot revealed high level of expression of LC3B-II in the MDS myeloid cells compared to controls indicating increased autophagic activity. The observed p62/SQSTM1 degradation along with the colocalization pattern of LC3B/LAMP-1 suggest increased autophagic flux. Metabolomic analysis of MDS myeloid lineage cells compared to controls revealed excessive glycolysis, defective oxidative phosphorylation and increased reductive carboxylation glutaminolysis associated with elevated level of intracellular 2-hydroxyglutarate, all indicative of HIF-1α driven metabolism. The co-localization between TOMM20 marker and autophagosomes in MDS myeloid cells was compatible with increased mitophagy whereas, MDS myeloid cells, were characterized by a reduction of mitochondrial mass and membrane potential in comparison to controls, as determined by FACS analysis. Conclusion: Our results provide evidence for the first time of the hypoxia-driven HIF-1α/REDD1/autophagy axis in the pathophysiology of MDS. Our study suggests that this deregulated pathway is responsible for the production of 2-hydroxyglutarate, an oncometabolite, which is implicated in dysregulated epigenetic homeostasis. All the above may lead to the dysregulated metabolism and differentiation potential of the myeloid cells, thus unraveling a new pathogenetic mechanism for the MDS development. Disclosures No relevant conflicts of interest to declare.