Frequency of BCR-ABL fusion transcripts in Serbian patients with chronic myeloid leukemia.

PURPOSE: The aim of this study was to analyze the occurrence of the most frequent BCR-ABL transcript variants (b3a2, b2a2 and e1a2) in Serbian patients with chronic myeloid leukemia (CML) and compare it with the occurrence reported in other populations. METHODS: We analyzed peripheral blood and bone marrow samples of 136 Serbian patients with CML by RT-PCR and cytogenetic methods. RESULTS: In 100 patients (73.5%) the b3a2 and in 34 (25%) the b2a2 forms of BCR-ABL were detected. One (0.75%) patient was BCR-ABL negative, but in lymphoblastic transformation he expressed the e1a2 [corrected] transcript of BCR-ABL. One (0.75%) patient displayed both b2a2 and b3a2 forms of BCR-ABL. Analysis of this group according to karyotype showed b3a2 predominance (79%) in patients with classic t(9;22); b2a2 was found in 20% and both b2a2 and b3a2 forms in 1%. In variant translocations b3a2 in 65% and b2a2 in 35% of the patients were detected. In contrast, the subgroup with normal karyotype expressed slight predominance of the b2a2 form (50%); b3a2 was found in 43% of the patients and one patient (7%) displayed e1a2. CONCLUSION: Predominance of the b3a2 form in Serbian patients with CML is in concordance with other relevant investigations, conducted mostly on Caucasian ethnic groups, but in contrast to the study performed on the Mestizo ethnic group in Ecuador. Slight predominance of the b2a2 form was also noticed among the patients with normal karyotype.

Trends in allogeneic haematopoietic cell transplantation for myelofibrosis in Europe between 1995 and 2018: a CMWP of EBMT retrospective analysis.

We performed a retrospective assessment of patient- and transplant-specific characteristics and outcomes for 4142 patients undergoing allogeneic haematopoietic cell transplant for myelofibrosis between 1995 and 2018 across 278 centres. Activity increased steadily across the four analysed eras (<2006, 2006-2010, 2011-2014 and 2015-2018). Median recipient age increased over time between the earliest and most recent cohort (49.4 years (range, 20.1-68) versus 59.3 years (range, 18.1-78.1). Increasing number of patients with a Karnofsky performance status <90 underwent transplant over time. Increased utilisation of matched unrelated donors was apparent (<2006, 22.5% versus 2015-18, 45.2%; p < 0.001). Decreased use of myeloablative conditioning, increased use of busulphan-based platforms and anti-thymocyte globulin was evident. Of note, rates of acute (a)GVHD grade II-IV by day +100 decreased over time (p = 0.027) as did rates of chronic (c) GVHD, predominantly extensive cGVHD (<2006, 36% (31-41%) versus 2015-18, 23% (21-25%); p = 0.001). Overall, significant factors associated with worse overall survival and non-relapse mortality (NRM) remained older age, use of donors other than matched sibling, recipient CMV seropositivity and a lower Karnofsky performance status (<90). Multivariable analysis demonstrated improvements in overall survival and reductions in relapse risk over time with stable NRM rates despite increasing numbers of older, less fit patients and use of unrelated donors.

Fine needle aspiration cytology in the diagnosis of head and neck masses: accuracy and diagnostic problems.

PURPOSE: Fine needle aspiration (FNA) cytology is an established technique associated with minimal complications compared with more invasive techniques such as wide core needle biopsy or open biopsy, and as such, very suitable for obtaining material in the delicate region of head and neck (H&N). The aim of this study was to assess the diagnostic accuracy of FNA cytology H&N masses. METHODS: Aspirations were performed by cytologists using 25 or 27G needles with 20 ml syringes attached, and smears were stained with May-Grunwald-Giemsa. Four hundred and ninety-four patients with palpable H&N masses underwent FNA during the study period of 2 years. RESULTS: Based on cytology alone, the most common findings were reactive lymphoid hyperplasia (28.5%), metastatic carcinoma (22.7%) and lymphoma (13.4%). Sixty-four (12.6%) FNA specimens were inadequate for diagnosis. Histological correlation was available in 164 (33.2%) patients who went on to have surgical excision of the mass. Nondiagnostic aspirate was in 16 (9.75%) patients, so the final group for cyto- histological correlation included 148 patients. The overall accuracy rate of FNA cytology, whether malignant or benign, was 91.89%, while the diagnostic accuracy for the exact type of tumor was 87.16%. There were 3 (2%) false-positive (FP) and 9 (6.1%) false-negative (FN) cytological diagnoses. The sensitivity and specificity of FNA cytology in determining a malignant diagnosis were 91.5% and 92.85%, respectively. Positive (PPV) and negative predictive value (NPV) were 97 and 81.25%, respectively. CONCLUSION: Our results showed that FNA cytology is a simple, safe, and cost-effective diagnostic method, suitable as a first-line investigation in palpable H&N masses. The main causes of the wrong diagnoses were sampling errors, inexperience and misinterpretation.

Expression of programmed cell death proteins in patients with chronic myeloid leukemia.

PURPOSE: Chronic myelogenous leukemia (CML) is a malignant myeloproliferative disease developing out of pluripotent hematopoietic stem cells that contain the fusion Bcr-Abl gene. The mechanisms that lead to these changes at molecular level are still unknown as are the mechanisms that increase the proliferative capacity of these cells. Disorders that occur in the process of apoptosis represent one of the possible molecular mechanisms that bring about disease progress. In our study we analyzed the presence of mutated (mut) p53 gene and the amplification of Bax proteins in patients with CML. PATIENTS AND METHODS: This study included 30 patients with CML (23 in chronic phase, 7 in blast transformation). Using immunohistochemistry with alkaline phosphatase / anti-alkaline phosphatase (APAAP) method we analyzed the expression of cell death proteins p53 and Bax in mononuclear bone marrow cells. Polymerase chain reaction single strand conformation polymorphism (PCR-SSCP) method was used to analyze the presence of mut p53 gene in mononuclear peripheral blood cells. Reverse transcription-polymerase chain reaction (RT-PCR) method was used to analyze the presence of Bcr-Abl in peripheral blood cells. RESULTS: High expression of Bax protein was detected in all analyzed patients, but no significant differences were noticed among them. No mut p53 gene was detected in any of the analyzed samples. Bcr-Abl b3a2 protein form was detected in all patients with variant translocations. CONCLUSION: Lack of mut p53 product in the peripheral blood and bone marrow cells in patients with CML suggests that this gene plays no important role in disease pathology. Increased level of Bax protein expression is an essential characteristic of CML cells but it is not related with the clinical stage of disease.

[Detection of K-ras and p-53 oncogene mutations in a patient with acute lymphoblastic leukemia before and after bone marrow transplantation]. / Detekcija mutacija u K-ras i p-53 onkogenima kod obolelog od akutne limfoblastne leukemije pre i posle transplanticije kostine srzi.

We present a case of 22 years old male patient, who was submitted to singenic transplantation of hematopoietic cells originating from the bone marrow in the remission phase of the diagnosed acute lymphoblastic leukemia (ALL). The bone marrow sample was donated by his healthy twin brother. The pretransplantation and transplantation phases were regular. We analyzed the presence of K-ras and p-53 point mutations in our patient with ALL and for the first time we had the opportunity to analyze the samples from two monozygotic twins. DNA was isolated from the peripheral blood mononuclear cells (PBMNC) by the standard procedure, of the patient with ALL before and after bone marrow (BM) transplantation and of his clinically healthy twin brother. Samples were subjected to PCR amplification of K-ras exons 1 and 2 and p-53 exons 5, 6, 7 and 8. In PBMNC of the patient with ALL before BM transplantation, mutations were observed in exon 1 of K-ras and exon 8 of p-53 gene. These mutations were found neither in PBMNC sample of his twin brother, nor in PBMNC of the patient with ALL after BM transplantation. In the p-53 exons 5, 6 and 7 and exon 2 of K-ras, there were no mutations in any analyzed samples. Detected mutations in K-ras and p-53 genes could be a part of larger genetic abnormalities and the obtained results had shown the possibility of using DNA mutational changes in the follow-up of the success of BM transplantation. The molecular disease marker that was found by this method was also significant for the detection of minimal residual disease at the molecular level.

[Treatment of Hodgkin's disease with high-dose chemotherapy and autologous stem cell transplantation]. / Lecenje Hodgkin-ove bolesti visokim dozama hemioterapeutike uz autologu transplantaciju maticnih celija hematopoeze.

High dose chemotherapy (HDC) with autologous stem cell transplantation (SCT) was applied for the treatment of 13 patients (pts) with Hodgkin's disease (HD) (10 with relapsed form and 3 with conventional chemotherapy resistant form) in the Clinic for Hematology, Military Medical Academy, from May 1997 to October 1999. After the initial treatment for the reduction of tumor, burden stem cells were mobilized by cyclophosphamide 2.5-7.0 g/m2 with G-CSF 5-12 micrograms/kg body mass (BM). The average number of colected mobilized mononuclear cells (MNC) was 2.99 (1.66-5.9) x 10(8)/kg BM by the apheresis large volume from peripheral blood. All patients received BEAM (BCNU, etoposide, Cyto-Ara, and melfalan) conditioning regimen with adequate supportive therapy. Good engraftment (100%) was observed at postransplantation period: number of polymorphonuclear cells was > 0.5 x 10(9)/l, on day 13th (10-21) and number of platelets > 20 x 10(9)/l, on day 17th (11-28). One patient (7.6%) died due to infective complications at day 98th after transplantation, 9 (69.2%) patients achieved complete and 3 (23.1%) patients partial remission of the disease. Out of three patients with partial remission, one relapsed, seven months after autologous SCT, with conventional chemotherapy resistant form and two, after the applied conventional locoregional radiotherapy reached remission. One patient (7.6%) developed secondary malignancy of acute myeloid leukemia form with threelinage displasy 27 months after autologous SCT. HDC with autologous SCT contributes to more successful treatment of early relapsed and standard chemotherapy resistant forms of HD and gives the opportunity for successful quality of living for those patients.

[Secondary myelodysplastic syndrome after autologous transplantation of hematopoietic stem cells in a female patient with Hodgkin's disease]. / Sekundarnih mijelodisplazni sindrom nakon autologe transplantacije maticnih celija hematopoeze kod bolesnice sa Hodgkin-ovom bolesti.

Secondary acute myeloid leukemia with threelineage displasy (sAML/MDS) has been described as a complication of therapeutic approach with high-dose chemotherapy and autologous stem cell transplantation (SCT) in the patients with Hodgkin's disease (HD). It is not yet clear whether the sAML/MDS is a consequence of a standard therapeutic regimen, applied before transplantation, or a high-dose chemotherapy. In a female patient with initially resistant form of HD at III-B-b clinical stadium (bulky disease in neck and mediastinum) after the initial treatment (MOPPx4; ABVDx3; BEA-COPPx2), high-dose chemotherapy has been applied according to BEAM protocol with autologous SCT. In a period after transplantation, radiotherapy (RT) has been applied at initial region of the disease and a patient reached complete remission (CR), which lasted for a 27 months. After that period sAML/MDS has been observed. Application of more standard therapeutic cycles and characteristic cytogenetic findings are the facts that support the opinion that sAML/MDS is a consequence of standard treatment before transplantation, rather than high-dose chemotherapy. That finding implies the need for correct choice of the HD patients suitable for early SCT therapeutic approach.

[Second allogenic bone marrow transplantation after late graft rejection in a patient with severe aplastic anemia]. / Sekundarna alogena transplantacija kostne srzi kod bolesnika sa teskom aplastickom anemijom nakon kasnog odbacivanja primarnog kalema.

Allogeneic bone marrow transplantation (BMT) is the treatment of choice in young patients (pts) with severe aplastic anemia (SAA) who have an HLA identical sibling donor. Late graft rejection to following allogeneic BMT for SAA is a significant clinical problem and is associated with a high risk of mortality. The optimal treatment strategy for patients with late graft rejection after first BMT is still an open question. We report 12-year-old patient with acquired SAA who underwent BMT from his HLA identical sister. BMT was first-line treatment within 3 months of diagnosis. Preparative therapy was Cyclophosphamide (Cy) 200 mg/kg body mass (BM) during 4 days. Graft versus host disease (GVHD) was prevented with Methotrexate (MTX), Methylprednisolone (MPDN) and Cyclosporin A (CsA). After 17 months, during which patient was with normal blood counts and full donor chimaerism, graft rejection occurred. The patient was re-engrafted from the same donor after conditioning with Cy 200 mg/kg BM plus horse antithymocyte globulin (ATG)--2 vials (á 25 mg)/10 kg BM over 4 days. Before the collection, donor's bone marrow was activated with low dose rhGM-CSF (3 micrograms/kg one day). Following a secondary BMT engraftment has sustained. The patient is alive with full donor chimaerism 26 months from secondary transplantation, without acute or chronic GVHD.

[Antilymphocyte globulin in the treatment of aplastic anemia]. / Antilimfocitni globulin u lecenju aplasticnih anemija.

Between January 1986 and July 1990, 17 patients with acquired aplastic anemia were treated with ALG or ATG combined with high doses of methylprednisolone. The mean age was 24.3 years (from 4 to 51 years). There were 9 cases with idiopathic etiology of acquired aplastic anemia; in 7 cases aplastic anemia was developed during or after HBsAg infection. In one case aplastic anemia was developed during tuberculous kidney infection. The remission of the disease was achieved in 11 of 17 cases (complete remission in 9-53%, and partial in 2-12% patients). Six (35%) patients did not respond to the treatment with ALG. One patient died of infection and hemorrhagic complications, two weeks after the therapy, without responding to the treatment with ALG. The four year survival rate without recidives was 65% (11/17). Four (23.5%) patients developed clonal diseases: PNH in 2; MDS in 1 and AL in 1 patient, 24, 38, 9 and 6 months after the therapy with ALG, respectively. The age of the patients is a valuable prognostic parameter (all patients under 20 years of age entered the remission), which cannot be said for pretreatment levels of reticulocytes, neutrophils and platelets. In none of the patients adverse effects of ALG were observed. The treatment was conducted in isolated rooms with sterile air circulation. ALG combined with high doses of methylprednisolone, for the majority of patients with aplastic anemia represents a drug of choice and is an appropriate alternative therapy to alogenic bone marrow transplantation, especially for patients with no HLA identical bone marrow donor.

Donor leukocyte infusion--the effect of mutual reactivity of donor's and recipient's peripheral blood mononuclear cells on hematopoietic progenitor cells growth.

Donor leukocyte infusions are an effective therapy for patients who relapse with leukemia after bone marrow transplantation. We report the case of 14-year-old boy who relapsed 34 months after sibling donor bone marrow transplant for Philadelphia-positive chronic myeloid leukemia. Subsequently, he received three infusions of donor mononuclear cells (DMNC) harvested in steady state hematopoiesis and one G-CSF mobilized-peripheral blood mononuclear cells (PBMC) infusion. Simultaneously, test named as--"Test of Mixed Progenitors" (TMP) was performed for the assessment whether the outcome of donor leukocyte infusion treatment could be predicted. Prior to DMNC infusions, the CFU-GM and BFU-E colony assays were performed for donor's and recipient's PBMC individually, as well as for the mixture of these cells at 1:1 ratio. The cells were plated either directly in the semisolid medium or after 24 h preincubation treatment. Significantly lower values for CFU-GM derived colonies were determined in TMP in comparison to the CFU-GM values obtained for the recipient's cells. The reduced number of CFU-GM was determined both in TMP performed without preincubation treatment, app. 80% and after the 24 h preincubation, app. 55%. The reduced number of BFU-E derived colonies (app. 44%) was observed only related to recipient's cells and after the preincubation treatment of the cells. The patient did not develop GVHD and currently (40 months after the first infusion). He remained well in complete hematological, cytogenetic, molecular and clinical remission, which was the most direct evidence of the GVL effect. The novel in vitro TMP test in which the specific contribution of donor's leukocytes to the growth of recipient's hematopoietic precursor cell growth was determined, correlated with the clinical outcome.