Panagiota Matsouka

Clinical relevance of balance between type 1 and type 2 immune responses of lymphocyte subpopulations in aplastic anaemia patients.

Immune dysfunction, which leads to the suppression of haemopoiesis by cytokines that are secreted by activated T lymphocytes, is considered to play a key role in the pathogenesis of acquired aplastic anaemia (AAA).

Alterations of Deadenylase Expression in Acute Leukemias: Evidence for Poly(A)-Specific Ribonuclease as a Potential Biomarker

Background/Aims: The degradation of mRNA is a key process in the control of gene expression correlated to anomalous cell proliferation. The rate-limiting step of mRNA degradation is the removal of the poly(A) tail by deadenylases. However, studies on deadenylase expression in cancer are limited. Herein, we analyzed the expression of several deadenylases from acute lymphoblastic leukemia (ALL) and acute myeloid leukemia (AML). Methods: Clinical samples from patients diagnosed with ALL and AML were the source of leukemic cells. Extracts from leukemic and control cells were analyzed for deadenylase mRNA levels using qRT-PCR, and the protein levels of PARN and CNOT7 deadenylases using immunoblotting. Results: RT-PCR analysis revealed altered expression for CNOT6, CNOT6L, CNOT7 and PARN deadenylases. The most significant alterations were observed for PARN and CNOT7 mRNA levels, which also reflect on the cognate protein level. Further analysis revealed that a significant amount of PARN is phosphorylated in ALL. Conclusions: We show that the expression of several deadenylases in acute leukemias is altered. The increase of PARN expression and the alteration of its phosphorylation status indicate important regulatory events. These data suggest that the role of deadenylases as auxiliary biomarkers and therapeutic targets should be meticulously investigated.

Oxidative stress due to radiation in CD34+ Hematopoietic progenitor cells: protection by IGF-1

Radiation exerts direct as well as indirect effects on DNA through the generation of reactive oxygen species (ROS). Irradiated hematopoietic progenitor cells (HPCs) experience DNA strand breaks, favoring genetic instability, due to ROS generation. Our aim was to study the effect of a range of radiation doses in HPCs and the possible protective mechanisms activated by insulin-like growth factor-1 (IGF-1). ROS generation was evaluated, in the presence or absence of IGF-1 in liquid cultures of human HPCs-CD34+ irradiated with 1-, 2- and 5-Gy X-rays, using a flow cytometry assay. Manganese superoxide dismutase (MnSOD) expression was studied by western blot analysis and visualized by an immunofluorescence assay. Apoptosis was estimated using the following assays: Annexin-V assay, DNA degradation assay, BCL-2/BAX mRNA and protein levels and caspase-9 protein immunofluorescence visualization. Viability and clonogenic potential were studied in irradiated HPCs. The generation of superoxide anion radicals at an early and a late time point was increased, while the hydrogen peroxide generation at a late time point was stable. IGF-1 presence further enhanced the radiation-induced increase of MnSOD at 24 h post irradiation. IGF-1 inhibited the mitochondria-mediated pathway of apoptosis by regulating the m-RNA and protein expression of BAX, BCL-2 and the BCL-2/BAX ratio and by decreasing caspase-9 protein expression. IGF-1 presence in culture media of irradiated cells restored the clonogenic capacity and the viability of HPCs as well. In conclusion, IGF-1 protects HPCs-CD34+ from radiation effects, by eliminating the oxidative microenvironment through the enhancement of MnSOD activation and by regulating the mitochondria-mediated pathway of apoptosis.

FIP1L1-PDGFRA molecular analysis in the differential diagnosis of eosinophilia

BackgroundPrimary eosinophlia associated with the FIP1L1-PDGFRA rearrangement represents a subset of chronic eosinophilic leukaemia (CEL) and affected patients are very sensitive to imatinib treatment. This study was undertaken in order to examine the prevalence and the associated clinicopathologic and genetic features of FIP1L1-PDGFRA rearrangement in a cohort of 15 adult patients presenting with profound eosinophilia (> 1.5 × 109/L).MethodsReverse transcriptase-polymerase chain reaction (RT-PCR) was used for the detection of FIP1L1-PDGFRA rearrangement and the results confirmed by direct sequencing. C-KIT-D816V mutation was analysed retrospectively by PCR and restriction-fragment-length-polymorphism (PCR-RFLP), in all cases with primary eosinophilia.ResultsTwo male patients with splenomegaly carried the FIP1L1-PDGFRA rearrangement, whilst 2 others were ultimately classified as suffering from idiopathic hypereosinophlic syndrome (HES) and one from systemic mastocytosis. These patients were negative for the C-KIT-D816V mutation and received imatinib (100–400 mg daily). Patients with CEL and HES responded to imatinib and remained in complete haematological, clinical and molecular (for carriers of FIP1L1-PDGFRA rearrangement) remission for a median of 28.2 months (range: 11–54), whilst the patient with systemic mastocytosis did not respond. Interestingly, in both patients with FIP1L1-PDGFRA rearrangement, the breakpoints into PDGFRA were located within exon 12 and fused with exons 8 and 8a of FIP1L1, respectively.ConclusionAn early diagnosis of FIPIL1-PDGFRA-positive CEL and imatinib treatment offer to the affected patients an excellent clinical therapeutic result, avoiding undesirable morbidity. Moreover, although the molecular mechanisms underlying disease pathogenesis remain to be determined, imatinib can be effective in patients with idiopathic HES.

The role of second-generation 5-HT3 receptor antagonists in managing chemotherapy-induced nausea and vomiting in hematological malignancies

Compared with solid tumor patients, those with hematological malignancies are at particular risk of chemotherapy-induced nausea and vomiting (CINV) because of their young age, exposure to highly-emetogenic induction, consolidation and salvage regimens, the high-dose conditioning regimens used before stem cell transplantation (SCT), and the heavy psychological burden of such treatments. In the absence of prophylaxis, around 75% of patients undergoing SCT experience delayed CINV. With first-generation 5-HT(3) receptor antagonists, only about 20% are completely protected from nausea and vomiting, and this frequent and debilitating adverse event has not been fully addressed. In contrast to solid tumors, there are no internationally agreed guidelines for the prevention and treatment of CINV in hematological malignancies. Work on a consensus is urgently required. The second-generation 5-HT(3) antagonist palonosetron is highly effective in preventing CINV in patients with solid tumors. The extended half-life of this agent and its mechanisms of action including allosteric binding, positive cooperativity and 5-HT(3) receptor internalization, may make it particularly effective in controlling delayed CINV. Although controlled comparisons against first-generation 5HT(3) agents have not yet been conducted in the setting of SCT, available evidence suggests that palonosetron may prove beneficial in preventing CINV in high risk patients with hematological malignancies.

Survivin isoform expression patterns in CML patients correlate with resistance to imatinib and progression, but do not trigger cytolytic responses

Tyrosine-kinase inhibitors are very effective in patients with CML, but in most cases the disease relapses after their discontinuation. As a result, novel approaches should be considered, such as anti-survivin treatment or anti-survivin-based immunotherapy. To gain insight into the roles of survivin isoform expression and specific CD8(+) T cells in CML, we investigated 51 patients at different stages, both at diagnosis and during treatment. We demonstrated that (i) patients at advanced-stage displayed an increased expression of the standard-survivin form along with a significant decrease of survivin-2B and -ΔEx3 levels, (ii) patients in chronic phase with higher expression of the standard-survivin exhibited a 3.5-fold increased probability not to achieve an optimal response to imatinib (p=0.048), (iii) responders displayed a significant up-regulation of all survivin isoforms in bone marrow, and (iv) anti-survivin CD8(+) T cells were undetectable both at diagnosis and during treatment. Accordingly, our results question the validity of immunotherapeutic approaches targeting survivin in CML.

Metabolism-Related Cytokine and Hormone Levels in the Serum of Patients with Myelodysplastic Syndromes

Background: A number of cytokines secreted from the bone marrow stromal cells and circulating hormones related to bone, adipose tissue and glucose metabolism might be involved in the pathogenesis of myelodysplastic syndromes (MDS). Methods: Serum levels of cytokines related to the metabolism of bone tissue [osteocalcin and parathyroid hormone (PTH)], adipose tissue (adiponectin, leptin and ghrelin) and glucose [insulin and insulin-like growth factor-1 (IGF-1)] were determined in 72 patients suffering from MDS, mostly of the low-risk group according to FAB classification, and 41 healthy individuals (controls). Results: Adiponectin and osteocalcin serum levels were significantly elevated in the MDS patients. Leptin, insulin and IGF-1 serum levels were reduced. No difference was found in the serum levels of PTH and ghrelin. Leptin levels were reversibly associated with patient blast count. Conclusion: Increased serum levels of adiponectin and low levels of IGF-1 in MDS patients may counterbalance the increased rate of apoptosis in the pool of hematopoietic progenitors. Osteocalcin secreted by osteoblasts regulates the renewal and proliferation of hematopoietic stem cells. Hormones and cytokines either secreted by the cells of the bone marrow stroma or transferred by the microcirculation act on hematopoietic progenitors and may regulate their differentiation, apoptosis and proliferation rate in MDS.

Simultaneous clinical resolution of focal segmental glomerulosclerosis associated with chronic lymphocytic leukaemia treated with fludarabine, cyclophosphamide and rituximab

BackgroundAlthough renal involvement in advanced haematological malignancies is common, glomerulonephritis associated with lymphoproliferative disorders is rare, and the related pathogenetic mechanisms are still poorly understood. We present a rare case of chronic lymphocytic leukaemia(CLL)-associated focal segmental glomerulosclerosis with nephrotic-range proteinuria.Case presentationA 53-year-old Caucasian man, previously healthy, with no history of hypertension, alcohol use or smoking presented with rapid weight gain, massive peripheral oedema, and hypertension. Laboratory findings included a white blood cell count of 49,800 cells/mm3 with an absolute lymphocyte count of 47,000 cells/mm3, serum albumin of 2.3 g/dL, urea 65 mg/dL, and creatinine 1.5 mg/dL. A 24-hour urine collection contained 7.1 g protein and significant haematuria. A peripheral blood smear showed mature lymphocytosis and smudge cells. Diagnostic imaging showed mild paraaortic lymphadenopathy with no renal abnormalities. Bone marrow aspiration and trephine biopsy showed diffuse and focal infiltration with B-CLL lymphocytes. Percutaneous renal biopsy revealed total sclerosis in 3/21(14%) of the glomeruli and focal and segmental solidification and sclerosis in 4/21 (19%) glomeruli. A regimen of fludarabine, cyclophosphamide and rituximab was successful in inducing remission of the CLL and clinical resolution of the nephritic-range proteinuria.ConclusionsA multidisciplinary approach to monitor both the malignancy and the glomerular lesions is crucial for the optimal management of paraneoplastic glomerulonephritis. Although chemotherapy with fludarabine, cyclophosphamide and rituximab successfully treated CLL-associated nephrotic syndrome in our patient, further studies are required to confirm efficacy in this setting.

Successful Control of Acute Myelofibrosis with Lenalidomide

Acute panmyelosis with myelofibrosis (APMF) is a rare, fatal hematological neoplasm that is characterized by the acute onset of cytopenias and fibrosis in the bone marrow in the absence of splenomegaly or fibrosis-related morphological changes in the RBCs. We present the case of a 59-year-old female who presented with a two-month history of anemia, leucopenia and a normal platelet count. The marrow was heavily fibrotic, and no aspirate material could be obtained; the biopsy showed extensive infiltration with small to medium size megakaryocytes, dysplastic changes in the erythroid compartment, and left shift in the myeloid cells. The patient was treated for four months with anabolic steroids (Danazol), growth factors and received regular blood transfusions. At 4 months after diagnosis, the patient was started on Lenalidomide, 10 mg/day for a 21-d-course along with growth factor support. At 6 months after treatment, the patient was transfusion-independent, had normalized blood counts, and, at 32 months on continuous lenalidomide treatment, her needs for growth factor support have been minimized. Repeat bone marrow biopsies showed a patchy distribution of fibrosis with areas of normal cellularity and morphology. To our knowledge, this is the first case for a medication that could reverse the fatal outcome of APMF.

Multiple autoimmune propensity and B-non-hodgkin lymphoma: cause or effect?

We report a case of multiple autoimmunity consisting of the presence of autoimmune haemolytic anaemia (AIHA), antimitochondrial antibodies (AMAs), and antiphospholipid antibodies (APLAbs) as the presenting manifestations of an extrahepatic B-non-Hodgkin lymphoma (B-NHL) in a 63-year-old woman. The patient presented with fatigue attributed to severe AIHA. Due to increased serum IgM and γ-GT levels, an investigation for AMA was performed, which proved positive with anti-M2 specificity. A prolongation of activated partial thromboplastin time (aPTT) led to the determination of APLAbs (lupus anticoagulant and other APLAbs) which were also positive. Bone marrow biopsy in combination with immmunohistochemical studies established the diagnosis of lymphoplasmacytic B-NHL. Ten months later, B-NHL was in remission while AMA and APLAbs were still positive. In conclusion, we documented the coexistence of multiple autoimmune reactions together with B-NHL highlighting the possible common pathogenetic pathways of the two entities.