Head and Neck Tumor Cells Exhibit Altered Proliferation upon Overexpression of nm23 Genes

nm23 was identified as a metastasis suppressor gene but is also appointed to a number of other biological functions. The goal of this study was to reveal the influence of ectopic expression of nm23-H1 and nm23-H2 on proliferation properties of head and neck tumor cells. The proliferation rate of transfected cells was evaluated using EGFP reporter system and flow cytometry. HEp-2 and CAL 33 cells transiently transfected with nm23 cDNA containing constructs exhibited enhanced proliferation. CAL 27 cells constitutively expressing GFP-Nm23-H2 protein, exhibited intense proliferation the first day after seeding, while the GFP-Nm23-H1 expressing clone started to proliferate after one-day lag period. The results on transiently transfected HEp-2 and CAL 33 cells generally confirmed previous findings connecting nm23 expression with altered proliferation of head and neck tumors. We speculate that the effects observed on stably transfected CAL 27 clones are due to their different attachment properties

CD20 Positive Childhood B-non Hodgkin Lymphoma (B-NHL): Morphology, Immunophenotype and a Novel Treatment Approach: A Single Center Experience

Lymphomas represent the third most common group of cancers in childhood and adolescence, mature B non Hodgkin’s lymphoma (B-NHL) accounting for up to 60% of newly diagnosed patients. The diagnosis of specific entities of B-NHL is based on well-defined morphologic analysis, immunophenotyping, cytogenetics and molecular genetics, which determine the optimal treatment strategy. In adult population a major turning point in treatment of B-NHL has been achieved since rituximab, in combination with CHOP has improved the survival rate up to 19%. Rituximab is a chimeric monoclonal antibody that targets CD20, a transmembrane calcium channel expressed on normal and malignant B-cells that mediates cytotoxic, apoptotic and anti-proliferative effects. The effect of rituximab in pediatric population is still not well enough investigated. Based on morphology and immunophenotype of malignant cells, seven children with B-NHL in our institution were eligible for treatment with modified B-NHL-Berlin-Frankfurt-Münster (BFM)-95-based protocol with rituximab administered on day -5. The complete remission was achieved in all seven patients. Six patients are still in complete remission at least 12 months after having finished chemotherapy and one patient relapsed two months after the last cycle and subsequently died. Major adverse effects observed during treatment were prolonged B-cell depletion and myelosupression. Rituximab in combination with B-NHL-BFM-95 protocol was otherwise well tolerated and proved to be effective in children and adolescents with B-NHL. The number of our patients is too small and the follow-up of a larger group of patients will help in defining the role of rituximab in the treatment of childhood B-NHL

The utilization of pEGFP reporter system in cell-cycle analysis of adherent cells

Background and Purpose: GFP (green fluorescent protein) is widely used in a variety of fluorescent methods aimed at revealing the fate of proteins in the cell, intracellular transport, transfection efficiency and is also recommended for cell-cycle analysis purposes. In our attempt to evaluate the role of nm23 genes in proliferation of head and neck tumor cells in culture we have decided to use EGFP reporter system and analyze the DNA content by flow cytometry. Materials and Methods: To optimize the method we either transiently transfected the cells with pEGFPC1-nm23 constructs or cotransfected the cells with an nm23 carrying constructs and pEGFPC1 as a reporter system. We established stable clones with pEGFPC1-nm23 constructs and analyzed them by flow cytometry, as well. Results and Conclusions:We report our experience for the use of pEGFP reporter system and flow cytometry for determining cell-cycle distribution of transiently and stably transfected adherent tumor cells. We discuss, in brief, the protocol we used and the problems that appeared during our experiments – GFP bleaching, cell clumping and degradation and insufficient number of cells to be analyzed. In conclusion, we suggest useful tips how to avoid or minimize the technical problems of this method and improve the results and analysis

Collection and composition of autologous peripheral blood stem cells graft in patients with acute myeloid leukemia: influence on hematopoietic recovery and outcome [Skupljanje i sastav transplantata autolognih krvotvornih matičnih stanica periferne krvi u bolesnika s akutnom mijeloičnom leukemijom: utjecaj na hematološki oporavak i ishod]

Hematopoietic stem cell (HSC) transplantation is a standard approach in the treatment of hematological malignant diseases. For the last 15 years the main source of cells for trasplantation have been peripheral blood stem cells (PBSC). With the availability of hematopoietic growth factors and understanding the advantages of treatment with PBSC, the application of bone marrow (BM) was supplanted. The aim of this survey was to explore the success of PBSC collection, the factors which influence the success of PBSC collection, the composition and the quality of graft and their infuence on hematopoietic recovery and outcome after transplantation in patients with acute myeloid leukemia (AML). PBSC were collected by the method of leukapheresis after applying a combination of chemotherapy and growth factors or only growth factors. The quality of graft was determined with the clonogenic progenitor cell assay and with the flow citometry analysis. Of the total 134 patients with AML, who were submitted to HSC mobilization, the collection was successful in 78 (58.2%) patients. The collection was more successful after the first than after the second attempt of HSC mobilization (49% vs. 11%). The criteria for effective mobilization were the number of leukocytes >3´109/L and the concentration of CD34+ cells >20´103/mL in the peripheral blood on the first day of leukapheresis. The number of CD34+ cells infused had the strongest impact on hematopoietic recovery. We noted significantly faster hematological recovery of neutrophils and platelets, fewer number of transfused units of red blood cells and platelets, shorter duration of the tranfusion support, shorter treatment with intravenous antibiotic therapy and shorter hospitalization after PBSC compared to BM transplantation. These advantages could provide their standard application in the treatment of patients with AML

Acute myeloid leukemia in children: the study of clinical and biological features and results of the treatment at the Department of pediatrics, Clinical Hospital Centre Rijeka

Cilj: Ispitati kliničke i biološke (citomorfologija, imunofenotipizacija, citogenetika) značajke te stopu preživljenja pedijatrijskih bolesnika s akutnom mijeloičnom leukemijom (AML) liječenih u tercijarnom centru. Ispitanici i Metode: Epidemiološko, retrospektivno, deskriptivno istraživanje u koje su uključeni bolesnici s novodijagnosticiranom AML mlađi od 18 godina, u razdoblju od 01.01.2002. do 31.12.2013. godine. Rezultati: U dvanaestogodišnjem razdoblju je hospitalizirano devetero djece s AML, 5 (55,6%) djevojčica i 4 (44,4%) dječaka. Prosječna dob je bila 11 (± 3,8 SD) godina. Najčešći simptomi i znaci su bili astenija (66,7%), perzistentna vrućica (55,6%), bljedoća (44,4%) i krvarenje (44,4%). Limfadenopatija i hepatomegalija su bile prisutne u 3 (33,3%), a splenomegalija u jednog (11,1%) bolesnika. Jedan (11,1%) bolesnik je imao inicijalno zahvaćen centralni nervni sustav. Trombocitopenija je bila prisutna u 8 (88,9%) bolesnika, anemija u 7 (77,8%) bolesnika, leukocitoza u 4 (44,5%) bolesnika, a leukopenija u 3 (33,3%) bolesnika. Svi bolesnici su imali blaste u razmazu periferne krvi. Utvrđeno je 7 citomorfoloških podtipova s karakterističnim imunofenotipskim značajkama. Sedam (77,8%) bolesnika je imalo numeričke i strukturne aberacije kromosoma. Tri (33,3%) bolesnika su svrstana u grupu standardnog rizika, a 6 (66,7%) u grupu visokog rizika. Remisija je postignuta petnaestog dana kemoterapije u 6 (66,7 %) bolesnika. Alogena transplantacija krvotvornih matičnih stanica je učinjena u 3 (33,3%) bolesnika. Osam (88,9%) bolesnika je u kompletnoj remisiji s medijanom praćenja od 10,1 godina. Smrtni ishod je nastupio u 1 (11,1%) bolesnika uslijed septičkog šoka. Zaključci: Pedijatrijska AML je heterogena hematološka neoplazma. Agresivna kemoterapija i transplantacija krvotvornih matičnih stanica omogućuju visoku stopu izlječenja.Objective: To investigate clinical and biological (cytomorphology, immunophenotyping, cytogenetics) features and the survival rate of pediatric patients with acute myeloid leukemia (AML) in a tertiary centre. Material and Methods: Epidemiological, retrospective, descriptive study involving patients with newly diagnosed AML younger than 18 years, from January 1st 2002 to December 31st 2013. Results: In a 12-year period, 9 children with AML were hospitalized, 5 (55.6%) girls and 4 (44.4%) boys. The median age was 11 (± 3.8 SD) years. The most common symptoms were asthenia (66.7%), persistent fever (55.6%), pallor (44.4%) and bleeding (44.4%). Lymphadenopathy and hepatomegaly were present in 3 patients (33.3%), and splenomegaly in one (11.1%) patient. One (11.1%) patient had initial central nervous system involvement. Thrombocytopenia was present in 8 (88.9%) patients, anemia in 7 (77.8%) patients, leukocytosis in 4 (44.5%) patients, and leukopenia in 3 (33.3%) patients. All patients had blasts in the peripheral blood smear. Seven cytomorphologic subtypes with characteristic immunophenotypic features were found. Seven (77.8%) patients had numeric and structural chromosome aberrations. Three (33.3%) patients were classified in standard-risk group and 6 (66.7%) in high-risk group. Remission was achieved in 6 (66.7%) patients on day 15 of chemotherapy. Allogeneic hematopoietic stem cell transplantation was performed in 3 (33.3%) patients. Eight (88.9%) patients are in complete remission with a median follow-up of 10.1 years. The death occurred in one (11.1%) patient due to septic shock. Conclusions: Pediatric AML is a heterogeneous hematologic neoplasm. Aggressive chemotherapy and hematopoietic stem cell transplantation provide a high cure rate

EXTRACORPOREAL PHOTOPHERESIS IN TREATMENT OF CHRONIC GRAFT VERSUS HOST DISEASE

Ekstrakorporalna fotofereza (EF) jest imunomodulatorna terapija koja se rabi u liječenju kronične reakcije transplantata protiv primatelja (engl. chronic graft versus host disease, cGVHD). Tijekom EF-a leukaferezom se iz krvi izdvajaju mononuklearne stanice, ex vivo im se dodaje 8-metoksipsoralen, stanice se ozrače UVA-zrakama i potom reinfundiraju bolesniku. Cilj istraživanja bio je procijeniti klinički i imunomodulatorni učinak postupka EF-a u bolesnika s cGVHD-om. Analiziran je 341 postupak EF-a u 7 bolesnika s cGVHD-om s medijanom EF-a po bolesniku 37 (raspon 13–131). U svih bolesnika cGVHD se manifestirao kožnim promjenama u kombinaciji sa simptomima drugih organskih sustava. EF su provođene dva dana za redom: prvih mjesec dana svaki tjedan, sljedeća 2 mjeseca svaki drugi tjedan, a potom jednom na mjesec. Medijan trajanja liječenja postupkom EF-a iznosio je 10 mjeseci (raspon 2 do 58). EF je većinom povoljno utjecao na simptome cGVHD-a pa je u 6 bolesnika došlo do poboljšanja i/ili stabilizacije promjena kože te bolje pokretljivosti zglobova, a u 2 bolesnika s ulceracijama sluznice usne šupljine promjene su se u cijelosti povukle. Do kliničkog poboljšanja došlo je 2 do 3 mjeseca nakon početka EF-a, što je omogućilo značajno smanjenje ili prestanak primjene glukokortikoida. Neželjene reakcije javile su se tijekom 4,9% postupaka. U bolesnika u kojih je došlo do poboljšanja kliničkog stanja normalizirale su se vrijednosti omjera CD4+/CD8+ stanica, kao i broj NK-stanica. Rezultati našeg istraživanja pokazuju da primjena EF-a povoljno utječe na simptome cGVHD-a i omogućuje sniženje doze kortikosteroida uz poboljšanje kvalitete života bolesnika pa se stoga može preporučiti za bolesnike koji ne odgovaraju na standardno liječenje.Extracorporeal photopheresis (ECP) is an immunomodulatory therapy which has been used in the treatment of chronic GVHD (cGVHD). ECP involves separation of the mononuclear cells with leukapheresis, followed by ex vivo administration of 8-methoxypsoralen and UV-A radiation and reinfusion to the patient. Aim of the study was to evaluate clinical and immunomodulatory effect of ECP procedures in patients with cGVHD. We analyzed 341 ECP procedures performed in 7 patients with cGVHD; median ECP per patient was 37 (range 13–131). All patients suffered from skin changes in combination with impaired joint mobility and symptoms of oral disease. ECP procedures were performed for two consecutive days: in initial phase weekly, followed by every two weeks and than monthly according to clinical response. Median of ECP treatment duration was 10 months (range 2–58). The effect of ECP in patients with cGVHD with skin and joint involvement was mostly beneficial: 6 patients experienced either improvement or stabilization in skin changes and joint mobility. In 2 patients who suffered from oral disease, the total recovery was observed. Clinical response was typically delayed until 2 to 3 months, and reduction in glucocorticoid dose was observed. Adverse reactions were observed in 4.9% procedures. In patients who responded to ECP treatment, CD4+/CD8+ ratio and number of NK cells were normalized. ECP proved to be an efficient and safe procedure that may be recommended for patients with cGVHD who do not respond to conventional therap

Collection and Composition of Autologous Peripheral Blood Stem Cells Graft in Patients with Acute Myeloid Leukemia: Influence on Hematopoietic Recovery and Outcome

Hematopoietic stem cell (HSC) transplantation is a standard approach in the treatment of hematological malignant diseases. For the last 15 years the main source of cells for trasplantation have been peripheral blood stem cells (PBSC). With the availability of hematopoietic growth factors and understanding the advantages of treatment with PBSC, the application of bone marrow (BM) was supplanted. The aim of this survey was to explore the success of PBSC collection, the factors which influence the success of PBSC collection, the composition and the quality of graft and their infuence on hematopoietic recovery and outcome after transplantation in patients with acute myeloid leukemia (AML). PBSC were collected by the method of leukapheresis after applying a combination of chemotherapy and growth factors or only growth factors. The quality of graft was determined with the clonogenic progenitor cell assay and with the flow citometry analysis. Of the total 134 patients with AML, who were submitted to HSC mobilization, the collection was successful in 78 (58.2%) patients. The collection was more successful after the first than after the second attempt of HSC mobilization (49% vs. 11%). The criteria for effective mobilization were the number of leukocytes >3´109/L and the concentration of CD34+ cells >20´103/mL in the peripheral blood on the first day of leukapheresis. The number of CD34+ cells infused had the strongest impact on hematopoietic recovery. We noted significantly faster hematological recovery of neutrophils and platelets, fewer number of transfused units of red blood cells and platelets, shorter duration of the tranfusion support, shorter treatment with intravenous antibiotic therapy and shorter hospitalization after PBSC compared to BM transplantation. These advantages could provide their standard application in the treatment of patients with AML

Subcutaneous Panniculitis-like T-cell Lymphoma in a 19 Month-old Boy: A Case Report

Subcutaneous panniculitis-like T-cell lymphoma (SPTCL) is a rare type of T-cell lymphoma of CD3+CD8+ phenotype characterized by deep-seated skin nodules or plaques mimicking panniculitis, a result of neoplastic lymphocytes infiltrating the subcutaneous fatty tissue. We present a case of a 19-month year old boy with SPTCL diagnosed and successfully treated in our institution. Disease first presented with symptoms of high fever and painful erythematous nodule located below the umbilicus. Later on the infiltrates appeared on the face, legs, arms and the back of the body. As the most decisive in obtaining the diagnosis, skin biopsy showed atypical, small to medium-sized lymphatic cells infiltrating the deeper dermal layers as well as the subcutaneous adipous tissue surrounding the adipocytes. Imunohystochemical analysis showed neoplastic lymphocytes positive for CD2, CD3, CD5, CD7, CD8, Tia-1, granzyme B and perforine, and negative for CD20, CD34, TDT and CD56. No infiltration of blood vessels or epidermis was evident. Specific T-cell lymphomas protocol (EURO-LB 02) was then initiated which resulted with rapid regression of all general and local symptoms. The treatment was completed according to schedule and the child is now, 24 months after the initiation of the treatment, in complete remission