Evaluation of the JAK2V617F Mutational Burden in Patients with Philadelphia Chromosome Negative Myeloproliferative Neoplasms: A Single-center Experience.

The identification of the JAK2V617F mutation in several distinct myeloproliferative neoplasms (MPNs) raised the question how one single mutation incites expression of at least three different clinical phenotypes, i.e., polycythemia vera (PV), essential thrombocythemia (ET) and primary myelofibrosis (PMF). In order to further evaluate already published data on the correlation between mutant JAK2V617F allele burden and specific hematological and clinical parameters, we tested the level of the JAK2 mutation in 134 JAK2+ patients with different MPNs. The patients were diagnosed according to the 2008 WHO criteria and followed for a median of 48 months. The JAK2 V617F quantification was done with a real time polymerase chain reaction (real time-PCR) method. The median allele burden was lowest in ET (25.8%), followed by 34.6% in PV and 51.8% in PMF patients (p<0.01). There was statistically significant association between the mutational load of 10.0-50.0% and blood count parameters in the PV patients (p<0.05). In PMF patients the mutational load was in correlation with older age and leukocyte count that were higher in patients with the mutational load of 10.0-50.0% and >50.0% compared to those with a mutational load of <10.0%. There were no statistically significant associations between the allele burden and blood counts in the ET cohort. Our study confirmed an association between the JAK2V617F allele burden and the distinct MPN phenotypes, indicating unfavorable prognosis in patients with a higher JAK2 allele burden. Our results suggest that JAK2 quantification should be incorporated in the diagnostic work-up of MPN patients as a useful tool for optimal treatment decision.

Single nucleotide polymorphisms of the inflamatory cytokine genes: interleukin-1B, tumor necrosis factors-A and tumor necrosis factor-B in adult patients with immune thrombocytopenia.

Immune thrombocytopenia (ITP) is an autoimmune disease characterized by thrombocytopenia due to platelet autoantibodies, causing an accelerated clearance of opsonized platelets by phagocytes. The etiology of ITP remains unclear, both genetic and environmental factors may have a role in the disease development. The aim of our study was to investigate a possible association of three single nucleotide polymorphisms (SNP) in the genes for interleukin beta (IL1B-511C/T), tumor necrosis factor beta (TNF+252G/A) and tumor necrosis factor alpha (TNFA-308G/A) with ITP. We have analyzed 125 adult patients with ITP and 120 healthy matched controls. Genotyping was performed by using PCR- RFLP methods. Our results demonstrated significantly different genotype distributions and allele frequencies for TNFB+252G/A in patients with ITP, p = 0.005 and p = 0.009 with Yates correction. We did not find any significant differences in the genotype distribution or allele frequencies for the other two genes. We have found significantly different genotype distribution and allele frequencies for TNFA-308G/A between patients with unresponsive and responsive ITP patients, p = 0.016 and p = 0.009. There were no significant differences in genotype distribution and allele frequencies for ILB-511C/T and TNFB+252G/A polymorphisms between those two groups of patients. We did not find any significant differences in genotype distribution and allele frequencies for all three polymorphisms between splenectomized and unsplenectomized ITP patients. The obtained data indicate that the A allele of TNFB+252G/A is more frequent in these patients than in the controls and that this polymorphism may play a significant role in disease susceptibility. The A allele of TNFA-308G/A was more frequent in patients with unresponsive ITP, indicating that this gene polymorphisms may contribute to therapy resistance.

Haematology in the Republic of Macedonia: present situation and brief history.

The development of clinical haematology in Macedonia has taken place over the past nine decades. The greatest expansion of its development took place in the second half of the 20th century. The oficial start of clinical haematology dates from 1956, when the Department of Haematology was founded within the framework of the Internal Medicine Clinic in Skopje. In the beginning, haematology represented a form of virtual sub-specialty, but its expansion was so progressive and rapid that it reached the highest peaks of Yugoslav haematology in those times. The period from 1968 to 1979 was a period of integral development of haematology and blood-transfusion science in Macedonia. Nowadays, the autonomous Public Health Institution, the University Hematology Clinic, is a unique healthcare, educational and scientific establishment in the Republic of Macedonia in its field of work. The diagnostics algorithm comprises cyto-morphologic and cyto-chemical analysis, through immunologic characterization with the assistance of a flow cytometer, to sophisticated molecular analysis for detecting genetic abnormalities. The therapeutic approach is based upon modern poly-haemotherapeutic protocols, application of monoclonal antibodies, immuno-modulatory agents, molecular target therapy and the use of alogeneic and autologous transplantation of fresh bone-marrow and frozen haemopoietic stem-cells. The current motto of the Haematology Clinic is: always help those who seek help, provide precise and early diagnostics, and apply all up-to-date therapeutic strategies, scientific research, continual education and day-to-day implementation of the latest achievements in the field of haematology in daily practice.

Association of polymorphisms in human platelet antigens with idiopathic thrombocytopenic purpura in Macedonians.

Idiopathic thrombocytopenic purpura (ITP) is an autoimmune disease characterized by thrombocytopenia due to the presence of platelet autoantibodies specific for platelet membrane glycoproteins, such as GPIIb/IIIa, GPIb/IX and GPIa/IIa. These autoantibodies cause an accelerated clearance of opsonized platelets by phagocytes and inhibition of platelet production. Human platelet antigen (HPA) systems HPA-1, HPA-2, HPA-3 and HPA-5 are components of platelet GP complexes GPIIb/IIIa, GPIb/IX and GPIa/IIa. The HPA system consists of more than 12 bi-allelic antigen polymorphisms in which a base-pair substitution leads to change in an amino acid sequence of a membrane glycoprotein expressed on the platelet surface. The aim of this study was to examine the association of HPA-1, HPA-2, HPA-3 and HPA-5 polymorphisms with idiopathic thrombocytopenic purpura. We performed genotyping of HPA-1, HPA-2, HPA-3, and HPA-5 systems in 60 patients with ITP and 120 healthy participants. Genotyping of HPA-1, -2, -3, and -5 alleles were performed by PCR and RFLP methods by using specific primers and restriction enzymes. Allele and genotype frequencies of HPA-1, HPA-3, and HPA-5 were not significantly different between patients and healthy participants. After Bonferroni adjustment a significant association in ITP patients with HPA-2 alleles (P=0.015, OR=1.923, CI=1.126-3.284) was found. Allele frequencies for HPA-2a were 0.852 in healthy participants and 0.750 in patients, and for HPA-2b 0.148 and 0.250 respectively. These results suggests that HPA-2b allele was more frequent in patients with ITP and may be involved in the formation of a specific autoepitope.

Assesment of non-relapse mortality (NRM) and long-term survival after autologous stem cell transplantation (ASCT) in patients with lymphoproliferative diseases.

A balanced risk-benefit approach to haematopoietic stem cell transplantation (HSCT) is the key for maximized chances of cure with acceptable quality of life for patients with advanced haematological malignancies. The aim was to try to assess the prognostic value of comorbidity and other independent variables concerning pretransplant mobilization strategies that affect auto SCT in patients with lymphoproliferative diseases.We stratified outcomes among 90 consecutive adult autologous recipients with lymphoproliferative diseases. 55% of patients were classified in the low index group for haematopoietic stem cell transplantation with comorbidity index (HCT-CI) scores between 0-1, 27% of patients with lymphoproliferative diseases had intermediate HCT-CI scores 1-2 and 12% of patients were in high HCT-CI group with a score≥3 and 6% undetermined. Two year NRM was 36% (95% CI: 17-36%), 26% (95% CI 15-39%) and 30% (95% CI: 22-39%) in the low, intermediate and high-risk HCT-CI groups respectively. The HCT-CI has been shown to sensitively capture organ comorbidities and provide valuable prognostic information for assignments of patients to clinical trials. Still, many questions remain to be answered, auch as good sample size, equal stratification of patients into risk groups, and also implementing better pretransplant assessment.

Immune thrombocytopenic purpura in adults in the last 10 years: single-centre experience.

BACKGROUND: Immune thrombocytopenic purpura (ITP) is a benign disease with low morbidity and mortality and frequent remissions that occur spontaneously or in response to first-line treatment with steroids or splenectomy. AIM: The purpose of this study is to describe the clinical outcomes of 170 patients with ITP diagnosed and/or treated in our hospital between 2000 and 2010. METHODS AND RESULTS: The median age at diagnosis was 47 years. Forty three (25%) were asymptomatic, 65% had minor skin or mucosal bleeding and 10% had significant bleeding from the gastrointestinal or genitourinary system. The median platelet count at diagnosis was 13x10(9)/L (range: 0-98x10(9)/L). Median follow-up of all patients was 13 months. Ninety-five patients had a follow-up longer than 12 months, with median 44 months (range 14-384). Corticosteroids were the initial treatment for 161/170 (95%) patients, 38 (22%) were splenectomized, 25 (14.7%) were treated with intravenous gamma globulins, while 9 did not received any specific treatment. A complete response to initial treatment (prednisone±splenectomy) was achieved in 55/161 (34.2%), a partial response in 90 (55.9%) and no response in 16 (9.9%) patients. In the group of patients with follow-up longer than 1 year; 28 (29%) patients had refractory or unresponsive ITP with a median follow-up of 66 months. All patients with refractory ITP were treated with steroids, 11 were splenctomized, significantly more patients with refractory ITP 12 (43%) were treated with IVIG compared with other ITP patients (16%), p=0.005. The median age of 38 splenectomized patients was 28 years and it is significantly different from the other patients (p<0.001). There were no significant differences in other characteristics between splenctomized or refractory ITP and other patients at diagnosis. CONCLUSION: Our results were similar to results already reported in other similar studies.

Minimal screening analysis based algorithm for diagnosis and clinical stratification of patients with acute myeloid leukaemia (AML): single centre experience.

In this paper we present our results from a study designed in order to establish and standardize a diagnostic algorithm for acute myeloid leukaemia (AML) in the Republic of Macedonia. A total of 146 consecutive adult patients (>15 years) were enrolled in the study. First, we determined the correct lineage assignment of the blast cells and evaluated the incidence of the favourable PML/RARα, AML1/ETO, CBFß/MYH11 genetic markers among the AML cases. Additionally, the obtained results were correlated with patients' age, comorbidities, and performance status, and each single AML patient was stratified to effective treatment strategy. Our results showed that morphology and cytochemistry established a lineage in 132 (89.1%) of the patients, but not in 16 cases that presented as acute leukaemia, of which 7 were assigned as myeloid, and in two a non-haematopoietic malignancy was indicated with immunophenotyping. Mulitparameter flow cytometry immunophenotyping also changed the assigned lineage based on morphology and cytochemistry in 5 (3.3%) of the patients from lymphoid to myeloid and improved diagnosis in 21 (14.1%) cases. By using a reverse transcriptase-polymerase chain reaction (RT-PCR) essay 28 (23.1%) patients were classified in the prognostically favourable AML genetic group; 8 patients expressed the fusion transcript PML/RARα AML1/ETO and 15 CBFß/MYH11. Moreover, analyses of the age, performance status and comorbidities further strtified an additional 12.5% of the patients to a different risk-adapted therapy. The applied minimal screening-analysis-based diagnostic algorithm enabled improved and more precise diagnosis and clinical stratification in 37.2 % of AML patients from our study group.

Immunosupressive treatment with cyclosporin-A for a patient with hypoplastic myelodisplastic syndrome: a case report.

BACKGRAOUND: Imunosupressive therapy with antithymocyte globulin (ATG), cyclosporine (CsA) or both has been shown to induce haematological responses in a subset of patients with myelodysplastic syndromes (MDS), in particular in the hypocellular form of MDS. CASE REPORT: We report our first case with hypocellular MDS treated with CsA. A 54-year-old female referred to our Department due to weakness and severe pancytopenia. Hypocellular form of MDS was diagnosed after bone marrow biopsy. Treatment with CsA was started one year after diagnosis. Treatment with CsA resulted in clinical improvement, a very good partial haematological response, resolution of transfusion requirement and an increase in bone marrow cellularity. CONCLUSIONS: In our experience, immunosuppressive treatment with CsA and/or ATG could be an alternative for patients with hypoplastic MDS for whom there is no possibility of allogenic bone marrow transplantation as only curative therapy.

Osteonecrosis of the jaw in patients with multiple myeloma treated with bisphosphanates.

Bisphosphonates are pyrophosphate analogues which inhibit osteoclastic activity. Long term use of bisphosphonates has recently been associated with osteonecrosis of the jaw (ONJ) defined as a three month non-healing defect in the jaw. ONJ is commonly precipitated by a tooth extraction or other stomatological procedure in patients treated with long-term, potent, high dose intravenous bisphosphonates for the management of myeloma, breast or prostate cancer. The aim of this study was to evaluate the incidence of ONJ in patients with MM treated with bisphosphonates during the last 8 years in our institution and to pre-sent the first two cases. We have analysed 247 myeloma patients diagnosed in our institution in the period 2002-09. Only 190/247 patients (76.9%) were treated with bisphosphonates. The incidence of ONJ in our group of patients treated with bisphosphonates was 2/190 (1%). The most commonly used bisphosponate was i.v. pamidronate (17.8%) and 46.6% were treated with two or more types of bisphosphonates. Sixty-five patients (34.2%) received oral forms of bisphosphonates; 42.1% patients were treated with i.v. forms of pamidronate, ibondronate or clodronate, and 45 patients (23.7%) received a combination of oral and i.v. forms of bisphosphonates. The mean duration of bisphosphonates therapy was 24.7±17.7 months. The low incidence of ONJ in our institution could be explained by the rare use of zolendronate, which is the most commonly referred bisphosphonate causing ONJ, and by a relatively shorter duration of bisphosphonates treatment in patients with MM. Despite the fact that ONJ is a rare complication in our institution, preventive measures must be considered.

A case report of aggressive adult neuroblastoma mimicking acute leukemia with fulminant course and fatal outcome.

A case of aggressive adult neuroblastoma mimicking acute leukemia with fulminant course and fatal outcome is described. Pancytopenia and circulating blasts cells at presentation suggested the diagnosis of acute leukemia in the previously healthy 38 years old Caucasian male patient, but flow-cytometry analysis of the bone marrow disclosed the correct diagnosis of neuroblastoma. The immunophenotype was CD45-/CD56+/CD9+ in around 50% of the mononuclear cells, indicating neuroectodermal origin of the malignant cells. Subsequently, the diagnosis was confirmed by immunohistochemical staining of a bone marrow biopsy. A review of the reported cases of neuroblastoma with leukemic features showed that several of them were misdiagnosed as having leukemia and that the diagnosis of neuroblastoma was made at autopsy examination, indicating that misdiagnosis may happen more often than is appreciated. It is in our opinion that the diagnosis of neuroblastoma should be considered in all cases of acute leukemia and pancytopenia, regardless of the age group of the patients.

JAK2V617F mutations in myeloid malignancies: single center experience.

Recently, V617F mutation in JAK2 tyrosine kinase gene was established as a marker of myeloproliferation, useful for proving clonality and securing diagnosis in a considerable proportion of the myeloproliferative neoplasms (MPN) The discovery presents a major breakthrough in the understanding of the pathogenesis of the MPN. Moreover, some studies suggest a possible role of the JAK2V617F mutation in the pathogenesis of some specific acute myeloid leukemia (AML) subtypes. To further improve the understanding of the role of JAK2V617F mutations in the pathogenesis and the clinical course of the myeloid malignancies we screened 192 patients with various MPN and AML for the mutations and analyzed the possible association between JAK2V617F mutations and the clinical features of MPNs patients. The frequency of V617F JAK2 mutation was analyzed by theallele-specific PCR assay. Out of 153 cases with known or suspected diagnoses of MPNs, 100 (65.3%) were positive for the JAK2V617F mutation and 53 (34.7%) were negative. In 39 AML cases the mutant allele V617F was not expressed. Correlations of the clinical features at diagnosis and long-term prognosis between the two JAK2-V617F different MPNs groups revealed comparability regarding all tested parameters except for the incidence of thrombotic history. Patients with the mutation had significantly higher incidence of thrombotic complication (38.5%), compared to the group without the mutation (19.2%) (P < 0.005). Our results confirmed the diagnostic significance of JAK2V617F mutation in MPNs and supported the notion that patients with the mutation should be classified in a new entity of MPNs.

Autologous stem-cell transplantation in patients with multiple myeloma.

BACKGROUND: - Multiple myeloma is a malignant plasma-cell proliferative disorder, the second most common haematologic cancer. Treatment with high-dose therapy (HDT) and single autologous stem cell transplantation (ASCT) is a category I recommendation of the National Comprehensive Cancer Network. Double transplantation can be proposed for patients failing to achieve small a, Cyrillic very good partial response (VGPR) after a first ASCT. Aims - The aim of this study is to analyse the effect of treatment with high-dose chemotherapy and autologous stem-cell support on survival in patients with multiple myeloma, and to compare our results with the results from other transplant centres. MATERIAL AND METHODS: - during a 7-year period we have performed 20 high-dose chemotherapy courses and autologous stem-cell transplantation on 17 patients (3 tandem transplantations) with multiple myeloma. In this trial we retrospectively analysed the epidemiology characteristics of these patients. Female: 9 Male - 8. Median age: 53 years (from 43-64 years). RESULTS: diagnosis was made according to Salmon and Durie criteria. High-dose regimen consisted of Melphalan doses of 200mg/m2. In tandem transplantations the dose of the second high-dose regimen was 140 mg/m2. The volume of CD34+ cells was approximately 3.8 x 10exp8/Kg.bw. In 3 patients we used phlebothomy as a source of added stem cells. The period from diagnosis to transplantation was 12 months. Of 17 patients 70% are alive, 5 have died (3 renal failure, 1 fatal cerebral bleeding and 1 with multiorgan failure). The disease-free survival was 22 months in our group of patients. Overall survival was 48 months and survival after transplantation was 35 months. The probability of 7 years' overall survival exists in 50% of patients. CONCLUSION: Patients treated with high-dose chemotherapy followed by autologous stem-cell support have a better survival and quality of life compared with patients treated with standard chemotherapy.

Stem cell transplantation - new treatment approaches.

Stem cell research still remains one of the most controversial fields of science today on account cell plasticity and its capability of transdifferentiation or de-differentiations to certain tissue types, as well as the clinical application of this scientific concept. Stem cells derived from bone marrow, peripheral blood or the umbilical cords are a common therapeutic approach for treatment of haematological malignancies as part of established transplant procedures (allogeneic, autologous, syngeneic stem cell transplantation). But recent clinical data have revealed the potential role of stem cells in the treatment of other nonhaematological diseases, degenerative disorders, cardiovascular diseases and autoimmune diseases. The experience with stem cell transplantation in haematological malignancies at the Hematology Department, Skopje, has been established since it was set up 7 years ago, with more than 130 patients undergoing transplant procedures (87 autologous and 43 allogeneic recipients). Encouraging results were also reported from the Skopje Cardiology Clinic in the field of intracoronary application of bone marrow derived stem cells for the treatment of patients with acute myocardial infarction. But this new rout in tissue regeneration should still be further extended and evaluated in clinically randomized studies that will confirm the therapeutic potential of stem cells.

Prognostic value of immunoglobulin variable heavy chain gene mutation status: long term follow-up in a series of chronic lymphocytic leukemia patients.

B-cell chronic lymphocytic leukemia (CLL) is a clinically heterogeneous disease, with many patients surviving for decades with minimal or no treatment, whereas others succumb rapidly to their disease despite therapy. Classical staging systems and laboratory features help predict survival in CLL, but they do not distinguish patients who will progress from those whose disease will remain indolent. In recent years, new molecular prognostic factors have emerged that have significantly improved prediction of the risk for disease progression. The mutational status of the immunoglobulin variable heavy chain genes (VH) is one of the major molecular prognostic factors. In this study we evaluated the association between the immunoglobulin VH gene mutation status and the clinical characteristics and outcome in 65 CLL patients that had been followed for a considerably long period at our institution. At diagnosis, patients with unmutated VH genes had higher median lymphocyte counts (P=0.001), higher total tumor mass score (P=0.001) and more often presented at an advance clinical stage (P=0.005) compared to patients utilizing mutated VH genes. Moreover, the median survival of patients with unmutated VH genes was considerably shorter (VH unmutated, 56 months, VH mutated, 125 months; P<0.001). These data confirmed the prognostic value of immunoglobulin VH genes mutational status in CLL, which divides the disease in two prognostic subsets in terms of overall survival and clinical characteristics of the disease. Analysis of the mutational status of the immunoglobulin VH genes may allow for an individualized approach to CLL treatment in the near future.

Restricted immunoglobulin VH region repertoire in chronic lymphocytic leukemia patients with autoimmune hemolytic anemia.

Between 10% and 25% of chronic lymphocytic leukemia (CLL) patients have episodes of autoimmune hemolytic anemia (AIHA) during the course of their disease. The anti-erythrocyte autoantibodies in most cases are polyclonal and express a different heavy chain isotype than the malignant clone, indicating that they are secreted by normal autoreactive B lymphocytes. To further investigate the pathogenesis of the AIHA in CLL, we analyzed the lg heavy (H) chain variable region genes expressed by leukemic cells from CLL patients with and without AIHA. Two VH genes were preferentially expressed by the leukemic cells in the CLL cases with AIHA and were present in 9 of the 12 investigated cases. The 51p1/DP-10 gene was expressed in 5 of these cases and was absent in the control group of 12 consecutive CLL cases without AIHA, whereas the DP-50 gene was present in 4 CLL-AIHA cases and only once in the control CLL group. A strikingly similar H-chain CDR3 region that contained a single reading frame of the DXP4 DH gene segment, and N-encoded proline at the DH/JH boundary, and a tyrosine-rich region encoded by the JH6 gene segment was observed in four CLL-AIHA cases. The preferential expression of two VH gene segments and a particular CDR3 region by the leukemic cells of patients with AIHA suggests that the antibodies produced by the CLL cells are directly involved in the pathogenesis of the hemolytic anemia.

Bone marrow transplants for paroxysmal nocturnal haemoglobinuria.

Paroxysmal nocturnal haemoglobinuria (PNH) is a rare clonal haematological disorder characterized by intravascular haemolysis and increased risk of thrombosis. PNH is associated with bone marrow failure syndromes including aplastic anaemia, myelodysplasia and leukaemia. Bone marrow transplants are sometimes used to treat PNH, but small series and reporting biases make assessment of transplant outcome difficult. The outcome of 57 consecutive allogeneic bone marrow transplants for PNH reported to the International Bone Marrow Transplant Registry (IBMTR) between 1978 and 1995 was analysed. The 2-year probability of survival in 48 recipients of HLA-identical sibling transplants was 56% (95% confidence interval 49-63%). Two recipients of identical twin transplants remain alive 8 and 12 years after treatment. One of seven recipients of alternative donor allogeneic transplants is alive 5 years after transplant. The most common causes of treatment failure were graft failure and infections. Our results indicate that bone marrow transplantation can restore normal bone marrow function in about 50% of PNH patients.