Higher percentage of in vitro apoptotic cells at time of diagnosis in patients with chronic lymphocytic leukemia indicate earlier treatment requirement: Ten years follow up

Introduction. Chronic lymphocytic leukemia (CLL) has an extremely variable clinical course. Biological reasons for that wide variation in clinical course and survival rates in CLL patients are not fully understood. Objective. The aim of the study was to evaluate the value of spontaneous apoptosis of CLL cells in vitro determined at presentation of disease, in prediction of treatment requirements and evolution of the CLL. Methods. Malignant B cells were isolated from the whole blood of 30 newly diagnosed CLL patients and cultured for 24 hours in RPMI-1640 medium supplemented with 10% of serum obtained from the same CLL patient. Cells were later fixed and processed for embedding in Epon, or cell smears were prepared and stained with TUNEL technique. Results. Ten-year follow-up revealed that patients with lower percentage of cells in apoptosis at presentation of disease had significant longer time treatment initiation (log rank test p<0.05). On the contrary, apoptosis of CLL cells was not shown to have significant impact on survival of patients (Kaplan Meier log rank test p>0.05). Conclusion. The results of this study emphasize the importance of apoptosis of CLL cells at the time of the initial diagnosis in pathobiology of this disease. [Projekat Ministarstva nauke Republike Srbije, br. 41025

Atypical pyoderma gangrenosum in a patient with osteomyelofibrosis

Background. Atypical forms of pyoderma gangrenosum generally appear on the upper extremities; most frequently they are associated with myeloproliferative disorders, including osteomyelofibrosis. A response to systemic steroids is more pronounced than in classical form. Sometimes it may be the first sign of an underlying malignancy. Case report. We reported a patient with atypical pyoderma gangrenosum developed during the course of a myeloid malignancy - osteomyelofibrosis. The lesions occurred after a minor trauma. Painful blistering plaques, with an elevated, bluish-gray border were located on the dorsal aspect of hands. No skin malignancy was found. The lesions resolved rapidly to systemic steroids. Conclusion. Considering the unusual clinical presentation which makes the diagnosis difficult, as well as the fact that atypical forms of pyoderma gangrenosum can be the first sign of malignancies, especially myeloproliferative ones, recognizing this entity enables timely guiding future investigations toward their prompt detection

The role of immunophenotyping in differential diagnosis of chronic lymphocytic leukemia

Introduction. Accurate diagnosis of chronic lymphocytic leukemia (CLL) acquires immunophenotyping by flow cytometry in order to facilitate differential diagnosis between CLL and other mature B-cell neoplasms (MBCN). Objective. The aim of this study was to define immunological profile of CLL cells. Methods. Immunophenotyping by flow cytometry was performed on peripheral blood specimens at diagnosis in the group of 211 patients with de novo MBCN. Results. Absolute count of B-cells was significantly increased in all MBCN patients comparing to healthy control group (p<0.05). B-cell monoclonality was detected in 96% of all MBCN patients, by using surface immunoglobulin (sIg) light chain restriction. B-cell antigens, CD19, CD20, CD22, were expressed with very high frequency in CLL and other MBCN. In comparison with other MBCN, in CLL group, the frequency of expression was higher for CD5 and CD23 (p<0.0001), though lower for FMC7 antigen (p<0.0001). CLL patients were characterized by lower expression patterns of CD20, CD22, CD79b, and sIg (p<0.0001) as well as higher expression pattern of CD5 antigen (p<0.05). Correlation between the final diagnosis of MBCN and values of CLL scoring system showed that the majority of CLL patients (97%) had higher values (5 or 4) whereas the majority of other MBCN patients (96%) had lower score values (0-3). Conclusion. Our results have shown that characteristic immunophenotype which differentiates CLL from other MBCN is defined by following marker combination - CD19+ CD20+low CD22+low CD5+high CD23+ FMC7- CD79b+low sIg+low. CLL score values of 5 or 4 points are highly suggestive for diagnosis of CLL

Brza progresija hronične limfocitne leukemije u Rihterov sindrom kod bolesnika sa kariotipom blizu triploidnog broja hromozom

Introduction. The presence of aneuploidy in patients diagnosed with chronic lymphocytic leukemia (CLL), except trisomy 12, is considered quite uncommon. Hyperdiploidy or near-tetraploidy (occurring in 1–3% of all CLL patients) usually confer a poor prognosis. Case report. We report a patient in a progressive phase of CLL with near–triploid karyotype. The prognosis of the disease was more precisely determined by applying the cytogenetic analysis of the karyotype and was complemented with molecular methods and pathohistological examination. The complex karyotype was accompanied by the TP53, C-MYC, and IGH gene disruptions, the most probable cause of rapid evolution into Richter’s syndrome. Conclusion. The use of comprehensive contemporary diagnostic techniques is highly recommended in patients who are in the progressive phase of CLL, primarily for the adequate choice of management strategy. The presented case confirms that aneuploidy in CLL patients indicates poor prognosis, which is in accordance with previous publications reporting on cases of CLL patients with aneuploidy.Uvod. Prisustvo aneuploidije kod bolesnika sa dijagnozom hronične limfocitne leukemije (HLL), sa izuzetkom trizomije 12, smatra se retkom pojavom. Pojava hiperdiploidnog ili kariotipa blizu tetraploidnog broja hromozoma (koji se javlja kod 1–3% svih bolesnika sa HLL) smatra se lošim prognostičkim parametrom. Prikaz bolesnika. Prikazan je bolesnik u uznapredovaloj fazi HLL sa kariotipom blizu triploidnog broja hromozoma. Prognoza bolesti je preciznije određena citogenetičkom analizom kariotipa bolesnika, i dopunjena molekularnim metodama i patohistološkom analizom. Otkriveno je prisustvo kompleksnog kariotipa udruženog sa poremećajima u genima TP53, C-MYC i IGH, što je najverovatnije bio uzrok brze progresije u Rihterov sindrom. Zaključak. Primena savremenih dijagnostičkih metoda veoma je značajna kod bolesnika u uznapredovaloj fazi HLL, prvenstveno zbog adekvatnog terapijskog pristupa. Prikazani slučaj ukazuje da je prisustvo aneuploidije kod bolesnika sa HLL loš prognostički znak, što je u saglasnosti sa prethodno publikovanim prikazima bolesnika sa HLL i sa aneuploidijom u kariotipu

Acute promyelocytic leukemia lacking t(15;17): Molecular evidence of atypical PML/RAR-α transcriptional variant by gene sequencing

Uvod. Precizno dijagnostikovanje akutne promijelocitne leukemije (APL), ne samo na osnovu morfoloških i kliničkih parametara, već i na molekularnom nivou, veoma je važno radi primene adekvatne ciljane terapije. Prikaz bolesnika. Prikazali smo bolesnicu, staru 62 godine, sa dijagnozom APL. Primenom standardne citogenetičke analize, kao i primenom fluorescentne in situ hibridizacije (FISH), nije bilo potvrđeno prisustvo t(15;17) kod opisane bolesnice. Primenom metode reverzna transkriptazalančana reakcija polimeraze (RT-PCR), identifikovana su dva atipična promyelotic leukemia/retinoic acid receptor alpha (PML/RAR-α) fuziona transkripta. Oba transkripta su predstavljala izoforme. Duži transkript je zadržao "okvir čitanja" i kodirao je funkcionalan PML/RAR-α aberantni protein, dok je kraći transkript bio van "okvira čitanja". Zaključak. Naša studija ukazuje na potrebu za primenom molekularne metodologije u svakodnevnoj kliničkoj praksi. Precizna karakterizacija PML/RAR-α fuzionih transkipta čini osnovu za identifikovanje retkih bolesnika čije lečenje zahteva dodatni oprez. Prema našim saznanjima, ovo je tek peti slučaj opisanog atipičnog PML/RAR-α transkripta koji u sebi sadrži celokupan PML egzon 7a, a među njima jedini koji se nije mogao detektovati primenom citogenetičke i FISH analize. Svi ovde predstavljeni slučajevi su imali smrtni ishod. Zbog toga, naši rezulatati, zajedno sa sličnim slučajevima opisanim u literaturi, naglašavaju značaj detaljne identifikacije atipičnih PML/RAR-α fuzija, ne samo u svrhu prepoznavanja njihove uloge u procesu leukemogeneze, veći i u smislu procene njihovog uticaja na ishod lečenja.Introduction. The accurate diagnosis of acute promyelocytic leukemia (APL), not only on the morphological and clinical, but also on the molecular level, is very important for application of targeted therapies. Case report. A 62year-old woman presented with APL. By using conventional cytogenetic analysis as well as applying the fluorescence in situ hybridization (FISH) analysis it has not been possible to confirm the presence of t(15;17) in the presented patient. Using reverse transcriptase polymerase chain reaction (RT-PCR) two atypical promyelotic leukemia/retinoic acid receptor alpha (PML/RAR-α) fusion transcripts were identified. Both detected transcripts were isoforms. The larger transcript was in-frame, coding for functional aberrant PML/RAR-α protein, while the shorter transcript was an out-of-frame. Conclusion. Our study highlights the need for the application of molecular methodology in daily clinical practice. Precise characterization of PML/RAR-α fusion transcript creates a basis for identifying rare individual cases that require special caution when treating such patients. To our knowledge this is only the fifth case of atypical PML/RAR-α transcript containing full PML exon 7a, and among them the only one that was cytogenetically cryptic and FISH negative. All of the herein presented cases had lethal outcome. Therefore, our findings with the additional review of the literature, emphasizes the importance of detailed identification of atypical PML/RAR-α fusions, not only for the purpose of knowing their role in leukemogenesis, but also for the assessment of the impact that they can have on the outcome of the treatment

Inflammation Promotes Oxidative and Nitrosative Stress in Chronic Myelogenous Leukemia

Chronic inflammation is characterized by the production of reactive oxygen species (ROS), reactive nitrogen species, and inflammatory cytokines in myeloproliferative neoplasms (MPNs). In addition to these parameters, the aim of this study was to analyze the influence of ROS on the pro-liferation-related AKT/mTOR signaling pathway and the relationship with inflammatory factors in chronic myelogenous leukemia (CML). The activity of the antioxidant enzymes superoxide dis-mutase, glutathione peroxidase, and catalase is reduced in erythrocytes while levels of the oxidative stress markers malondialdehyde and protein carbonyl are elevated in the plasma of patients with CML. In addition, nitrogen species (nitrotyrosine, iNOS, eNOS) and inflammation markers (IL-6, NFkB, and S100 protein) were increased in granulocytes of CML while anti-inflammatory levels of IL-10 were decreased in plasma. CML granulocytes exhibited greater resistance to cytotoxic H2O2 activity compared to healthy subjects. Moreover, phosphorylation of the apoptotic p53 protein was reduced while the activity of the AKT/mTOR signaling pathway was increased, which was further enhanced by oxidative stress (H2O2) in granulocytes and erythroleukemic K562 cells. IL-6 caused oxidative stress and DNA damage that was mitigated using antioxidant or inhibition of inflammatory NFkB transcription factor in K562 cells. We demonstrated the presence of oxidative and ni-trosative stress in CML, with the former mediated by AKT/mTOR signaling and stimulated by in-flammation

Macrophages Provide Essential Support for Erythropoiesis, and Extracellular ATP Contributes to a Erythropoiesis-Supportive Microenvironment during Repeated Psychological Stress

Psychological stress is a significant contributor to various chronic diseases and affects multiple physiological processes including erythropoiesis. This study aimed to examine the tissue-specific contributions of macrophages and extracellular ATP, as a signal of disturbed tissue homeostasis, to erythropoiesis under conditions of repeated psychological stress. Adult male BALB/c mice were subjected to 2 h daily restraint stress for seven consecutive days. Clodronate-liposomes were used to deplete resident macrophages from the bone marrow and spleen two days prior to the first restraint procedure, as well as newly recruited macrophages, every third day for the duration of the experiment. Repeated stress induced a considerable increase in the number of erythroid progenitor cells as well as in the percentage of CD71+/Ter119+ and CD71−/Ter119+ cells in the bone marrow and spleen. Macrophage depletion completely abolished the stimulative effect of repeated stress on immature erythroid cells, and prevented stress-induced increases in ATP levels, P2X7 receptor (P2X7R) expression, and ectonucleotidase CD39 activity and expression in the bone marrow and spleen. The obtained results demonstrate the stimulative effects of repeated stress on erythroid cells, extracellular ATP levels, P2X7R expression, CD39 activity and expression within the bone marrow and spleen, as well as the essential role of macrophages in stress-induced changes

Myositis-specific autoantibodies in a non-traveler, patient from a non-endemic country, with Plasmodium vivax malaria

Introduction: Autoantibodies (AAb) are a hallmark of immune-mediated inflammatory diseases. Malaria is a parasitic disease caused by Plasmodium protozoa. Individuals with malaria may present with a wide range of symptoms. It is frequently linked to the development of different AAb.Case description: A 35-year-old male presented with repeated episodes of fever, malaise, myalgia, dark urine, and yellowish sclera. Initial diagnostic workup revealed severe Coombs-positive anemia, increased C-reactive protein, and procalcitonin, pathological liver tests, high concentration of serum IgE, IgG, IgM, IgA, positive antinuclear antibodies (ANA), and positive antineutrophil cytoplasmatic antibodies (ANCA). In addition, myositis-specific antibodies directed to polymiositis-scleroderma 75 protein (PmScl75), threonyl-tRNA synthetase (PL-7), alanyl-tRNA synthetase (PL-12), Mi-2 antigen (Mi-2), Ku DNA helicase complex (Ku), signal recognition particle (SRP), and antiaminoacyl tRNA synthetase (EJ) were detected. The patient was suspected of having systemic lupus erythematosus and sent to the Clinic of Allergy and Immunology for further evaluation and treatment. A peripheral blood film examined by the hematologist during an episode of fever revealed intra-erythrocytic parasitic forms of Plasmodium vivax (P. vivax). After being diagnosed with P. vivax malaria, he was transferred to the Clinic for Infective and Tropical Diseases. The therapy consisted of artesunate/mefloquine and prednisone led to a complete clinical recovery and autoantibodies gradually disappeared.Conclusions: Malaria would not normally be considered during the initial diagnostic workup in a non-traveler and a patient from a non-endemic country. However, a thorough parasitic evaluation in patients presenting with a broad range of autoantibodies might be of particular importance

Easily Applicable Predictive Score for Differential Diagnosis of Prefibrotic Primary Myelofibrosis from Essential Thrombocythemia

Essential thrombocythemia (ET) and prefibrotic primary myelofibrosis (prePMF) initially have a similar phenotypic presentation with thrombocytosis. The aim of our study was to determine significant clinical-laboratory parameters at presentation to differentiate prePMF from ET as well as to develop and validate a predictive diagnostic prePMF model. This retrospective study included 464 patients divided into ET (289 pts) and prePMF (175 pts) groups. The model was built using data from a development cohort (229 pts; 143 ET, 86 prePMF), which was then tested in an internal validation cohort (235 pts; 146 ET, 89 prePMF). The most important prePMF predictors in the multivariate logistic model were age ≥ 60 years (RR = 2.2), splenomegaly (RR = 13.2), and increased lactat-dehidrogenase (RR = 2.8). Risk scores were assigned according to derived relative risk (RR) for age ≥ 60 years (1 point), splenomegaly (2 points), and increased lactat-dehidrogenase (1 point). Positive predictive value (PPV) for pre-PMF diagnosis with a score of ≥points was 69.8%, while for a score of ≥3 it was 88.2%. Diagnostic performance had similar values in the validation cohort. In MPN patients with thrombocytosis at presentation, the application of the new model enables differentiation of pre-PMF from ET, which is clinically relevant considering that these diseases have different prognoses and treatments

Clinical significance of optimal red cell mass and plasma volume estimation methods

BACKGROUND: The aim of this study was to present and compare the results of proposed methods for optimal red cell mass and plasma volume (RCM&amp;PV) estimation, and their influence on the interpretation of obtained results. MATERIAL AND METHODS: In 120/280 patients with polycythaemia rubra vera, subjected to RCM&amp;PV determination with autologous erythrocytes in vitro labelled with 51Cr-sodium chromate, optimal volumes were determined using: 1. traditional ml/kg of: - the real body weight method (ml/kg RBW); - the optimal body weight method (ml/kg OBW). 2. the body weight, height, and sex based method (Retzlaff's tables), 3. the method recommended by the International Council for Standardization in Haematology (ICSH), based on body surface area. RESULTS: Different interpretation of the same results of 120 RCM&amp;PV measurements was registered in 48/120 patients (40%). The greatest disagreement existed between ml/kg RBW and ml/kg OBW methods (in 39/120 subjects, 32.5%). In underweight patients the ml/kg RBW method, and in overweight patients the ml/kg OBW method, offered better agreement with ICSH&amp;Retzlaff's methods. The ml/kg RBW method disagreed with ICSH&amp;Retzlaff's methods and ml/kg OBW in 25% and 19.2% of patients respectively. ICSH and Retzlaff's methods disagreed in 10/120 patients (8.3%). The ICSH method yielded significantly lower optimal volumes than Retzlaff's. CONCLUSION: Three methods for optimal RCM&amp;PV estimation lead to different interpretations of the same results of RCM&amp;PV measurements with 51Cr-erythrocytes in 40% of patients. Two ml/kg body weight methods show greater disagreement in comparison with ICSH and Retzlaff's methods, which differ significantly. The ICSH method yields lower optimal values compared to Retzlaff's