A Case of Acute Myeloid Leukemia with Novel Translocation t(6;11)(p22.2;q23) and Concurrent Insertion ins(11;9)(q23;p21.3p21.3).

We describe the case of a boy with acute myeloid leukemia with translocation t(6;11)(p22.2;q23) and insertion ins(11;9)(q23;p21.3p21.3). Translocation t(6;11)(p22.2;q23) involving the short arm of chromosome 6 has not been previously described. The LDI-PCR showed the presence of KMT2A-MLLT3 fusion and identified the BTN3A1 (butyrophilin subfamily 3 member A1) gene on 6p22.2 as the other KMT2A translocation partner. The BTN3A1 gene has never been described in the context of acute leukemia. Although this fusion is out of frame, as the antisense strand of BTN3A1 is fused to the sense strand of KMT2A, the loss of heterozygosity of the BTN3A1 gene might contribute to the malignancy of leukemic cells.

Impairment of Immune Function in Children with Familial Hemophagocytic Lymphohistiocytosis.

Hemophagocytic lymphohistiocytosis (HLH) is a severe systemic syndrome associated with hyperactivation of macrophages and impaired regulation of the immune system. Two forms of HLH are currently recognized: genetically determined or familial (FHLH), and secondarily developed in the course of primary diseases, like autoimmune disorders, rheumatoid disorders, cancers, or infections. In the Polish population, FHLH is rather rare. The aim of the present study was to assess the immune function in a group of children with clinical symptoms suggesting FHLH. Forty five children with suspected HLH of the median age of 4 years and 15 healthy children, taken as a control group, were enrolled into the study. All presented results were obtained with the use of flow cytometry. In the HLH group, there were only three cases identified with the UNC13D gene mutation responsible for the FHLH3 phenotype. Another four children, without known mutation, were classified as FHLH because of frequent recurrence of the disease. In all cases of FHLH, cell cytotoxicity was impaired compared with healthy children (p = 0.003). Perforin expression in FHLH was normal or higher than that observed in controls (p = 0.09). In case of patients with mutation in the Munc13 protein, degranulation was lower than that in healthy children (<5 %). The findings of this study demonstrate that children with known mutations responsible for the FHLH development are immunocompromised. However, it requires further elucidation whether the presence of currently unknown mutations could lead to a similar phenotype.

The immunophenotypes of blast cells in B-cell precursor acute lymphoblastic leukemia: how different are they from their normal counterparts?

BACKGROUND: Currently, there are three major maturational stages of CD19 antigen expressing B-cell precursors (hematogones). In B-cell precursor acute lymphoblastic leukemia (BCP-ALL), the malignant counterpart of hematogones, the leukemic blasts share common phenotypic features. The aim of the study was to enumerate the actual differences between the leukemic blasts in the CD10+ and CD10- subgroups of BCP-ALL and hematogones by assessing the expression of the antigens: TdT, CD34, CD45, CD10, CD38, CD20 and CD22. METHODS: To enable quantitative assessment of antigen expression on the different cell types, an objective scale of antigen expression was developed, the basis of which was direct fluorescence measurement using multicolor flow cytometry. RESULTS: All cases of CD10+ BCP-ALL clustered with type 1 hematogones. Among CD10-- BCP-ALL subgroup, 54.5%, 27.3% and 18.2% of cases clustered with type 1, 2 and 3 hematogones, respectively. In contrast to the CD10- blasts, the CD10+ blasts exhibited significantly higher levels of TdT, CD22, CD34 and CD20 expression. Conversely, CD10- blasts showed significantly higher expression of CD45 than CD10+ blasts, and a higher rate of CD45 antigen overexpression than CD10+ blasts (54.5% vs. 14.9% of cases, respectively). CONCLUSIONS: Multiparameter flow cytometry combined with the use of absolute antigen expression scale based on direct fluorescence measurement, has enabled a clear distinction between blasts in BCP-ALL cases and their normal counterparts. This novel and previously undescribed method has allowed the comparative analysis of antigen expression between leukemic blasts and different types of their normal counterparts.

Tsc1-Tp53 loss induces mesothelioma in mice, and evidence for this mechanism in human mesothelioma.

Mesothelioma is diagnosed in ∼2500 patients in the United States every year, most often arising in the pleural space, but also occurring as primary peritoneal mesothelioma. The vast majority of patients with mesothelioma die of their disease within 3 years. We developed a new mouse model of mesothelioma by bladder or intraperitoneal injection of adenovirus Cre into mice with conditional alleles of each of Tp53 and Tsc1. Such mice began to develop malignant ascites about 6 months after injection, which was due to peritoneal mesothelioma, on the basis of tumor morphology and immunohistochemical staining. Mesothelioma cell lines were established, which showed loss of both Tsc1 and Tp53, with mammalian target of rapamycin complex (mTORC)1 activation. Treatment of mice with malignant ascites due to mesothelioma with rapamycin led to a marked reduction in ascites, extended survival and a 95-99% reduction in the mesothelioma tumor volume, in comparison with vehicle-treated mice. To see whether TSC1/TSC2 loss was a common genetic event in human mesothelioma, we examined nine human mesothelioma cell lines, and found that four of nine showed persistent activation of mTORC1, although none had loss of TSC1 or TSC2. A tissue microarray analysis of 198 human mesothelioma specimens showed that 33% of cases had reduced TSC2 expression and 60% showed activation of mTOR, indicating that mTOR activation is common in human mesothelioma, suggesting that it is a potential therapeutic target.

Flow cytometry in detection of abnormalities of natural killer cell.

The population of natural killer (NK) cells is very heterogeneous and plays a role in the immune system. Several NK cells subpopulations are recognized, differing in phenotype, cytokine release and cytotoxic ability. Different expression of biologically relevant molecules on the surface of NK cells may indicate their multiple functions. The activity of NK cells has mainly to do with their cytotoxic nature. A complete analysis of NK cells function requires application of many tests because a defect may be present at different stages of the cytotoxic process, from signal transduction through lysosome degranulation to target cells destruction. Flow cytometry is actually one of the best methods for the identification of NK cells and tracking their defects.

Acute lymphoblastic leukaemia cells express CCR7 but not higher amounts of IL-10 after CD40 ligation.

OBJECTIVE: Production of cytokines that support T-cell activation and proliferation and migration to lymph nodes is one of the most important terms of cancer vaccine development. In previous studies we and others used CD40 ligation to obtain higher expression of co-stimulatory and adhesion molecules on leukaemic cells from children with acute lymphoblastic leukaemia (ALL). This time we assess the cytokine and chemokine gene expression profile in CD40-stimulated ALL cells. MATERIAL AND METHODS: Malignant cells from 25 children with BCP-ALL were stimulated (or not) with huCD40LT and rIL-4 for 96 h. Eleven different molecule, cytokine and chemokine mRNAs levels (CCR7, IL-23, TGF-beta-IP, IFN-gamma, IL-10, CD1a, CD40, CD54, CD80, CD83, CD86) were determined using the real-time PCR technique with TaqMan chemistry using ready-to-use low-density arrays for gene expression by Applied Biosystems. RESULTS: 1) Increases in mRNA levels for CD40, CD54 and CD80 after CD40L and IL-4 stimulation were observed, 2) CCR7 mRNA expression was higher after CD40 ligation than before the culture (p = 0.002), 3) IL-10 mRNA expression was higher after the culture with medium than before the culture (p = 0.01). CONCLUSIONS: The results show that leukaemia-derived dendritic cells obtained with CD40 ligation express CCR7 - chemokine is involved in migration to lymph nodes and does not produce higher amounts of IL-10, a potent immunosuppressive cytokine. Our preclinical findings could be used in the design of immunotherapy trials for the treatment of children with ALL.

Low expression of costimulatory molecules and mRNA for cytokines are important mechanisms of immunosuppression in acute lymphoblastic leukemia in children?

UNLABELLED: Mechanisms leading blasts of acute lymphoblastic leukemia to escape from immune surveillance are still unknown. Only few reports showed that ALL cells are inefficient antigen presenting cells. The aim of the study was to assess expression of critical costimulatory/adhesion molecules and mRNA for main pro- and anti-inflammatory cytokines in ALL cells. Children with B-cell precursor ALL (n=20) were prospectively enrolled into the study. Expression of costimulatory/adhesion molecules (CD1a, CD11c, CD40, CD54, CD80, CD83, CD86, CD123, HLA class I and II) was assessed by flow cytometry and mRNA for cytokines (IFN-gamma, IL-10, IL-4, TGF-beta) - with real-time PCR. RESULTS: 1) high expression was observed for HLA I and II class, moderate for CD40, CD83, CD86 and low or no expression for CD80, CD54, CD1a, CD11c and CD123; 2) we found expression of mRNA for IFN-gamma, IL-10, IL-4 and TGF-beta in blasts cells (but not in all specimens). We noted relatively lower expression of all assessed cytokines comparing to T-cells obtained from healthy donors but interestingly expression for IL-10 was higher in normal B-cells than in blast cells, and IFN-gamma and IL-4 were not found in normal B-cells. In summary we suggest that ALL-blasts present low expression of costimulatory/adhesion molecules and mRNA for cytokines and this probably contribute to the absence of host T- cells stimulation to immune response.

[Long-term results of stapedectomy in pediatric patients]. / Odlegle wyniki stapedektomii wykonywanych u dzieci.

Otosclerosis is generally considered as a disease of adults, but the onset of hearing loss can occur in childhood. The purpose of this presentation is to show the effectiveness of stapedectomy in children. Both, short and long-term results are presented. Material is based on 14 pediatric patients, age 7-14 y. 12 of them had bilateral, 2 unilateral hearing loss. Together 26 ears. The diagnostic pattern was as follow: general pediatric and ENT examination, tuning fork testing, pure-tone audiometry (2x), recognition score test, tympanogram and acoustic reflex test, CT scan of the temporal bone. Particular attention was paid to the parents report concerning the episodes of otitis media or frequent upper respiratory tract infections and history of family hearing loss. Average time of hearing loss was noted 3 years before stapedectomy. Pure tone audiometry was performed on frequencies 500, 1000, 2000, 3000 and 4000 Hz. All children had hearing loss with air-bone gap 30 or more dB. 23 ears underwent stapedectomy for otosclerosis with teflon-piston prosthesis insert. Hearing level was based on PTA obtained in 2-3 weeks postoperatively and then 3 and 6 months. PTA hearing improvement was in 22 ears. No hearing loss was noted after operation. A total of 16 ears were available for long-term follow-up (up to 24 months after stapedectomy). The PTA hearing improvement was found to be permanent.

In vitro sensitivity of leukemic cells to nucleoside derivatives in childhood acute leukemias: good activity in leukemic relapses.

Nucleoside analogues such as fludarabine and cladribine are used in therapy of indolent lymphomas and leukemias in adults, while cytarabine is used mainly in protocols for acute leukemias. Mechanisms of their activity is based on inhibition of enzymes involved in DNA, RNA and protein synthesis. The objective of the study was the analysis of in vitro cellular drug sensitivity in childhood acute lymphoblastic (ALL) and myeloid (AML) leukemia. Isolated leukemic cells obtained from 264 patients, including 152 initial ALL, 45 relapsed ALL, 54 initial AML and 13 relapsed AML were tested for cytotoxicity for fludarabine, cladribine, and cytarabine by the MTT assay. Drug concentration lethal to 50% of tested cells was regarded as a value of drug resistance. Three tested nucleoside analogues showed highest cytotoxicity against initial ALL samples. Samples of relapsed ALL and initial AML were more resistant than ALL de novo ones. Unexpectedly, no differences were observed between initial and relapsed AML samples for all tested drugs, what suggests that nucleoside analogues are active drugs in relapsed AML, which is commonly regarded as a resistant disease. All tested drugs presented significant cross-resistance in each of analyzed subgroups. In summary, tested nucleoside analogues presented relatively good activity against childhood leukemias at relapse stage.

mRNA for chosen pro- and anti-inflammatory cytokines in T-lymphocytes in paediatric leukemias and lymphomas--a preliminary report.

We assessed mRNA for chosen pro- and anti-inflammatory cytokines in T-lymphocytes of peripheral blood in paediatric patients with leukemias and lymphomas. Levels of four different cytokine mRNAs (IFN-gamma, IL-10, IL-4, TGF-beta) were determined by the real-time PCR technique. In the whole examined group, at the time of diagnosis, we noted lower amounts of mRNA for TGF-beta1, comparing to respective values in the control patients. In the ALL group, we observed the following: 1) at the time of diagnosis: lower amounts of mRNA for IL-4 and for TGF-beta1, comparing to respective values in the control group; 2) lower amounts of mRNA for IL-10 after remission induction, comparing to the time of diagnosis. In our opinion, "immunedysregulation" in lymphoproliferative diseases in children is not caused by IFN-gamma deficiency. The deficit of anti-inflammatory cytokines, i.e., IL-4, TGF-beta, with higher amounts of IL-10, suggests their role in cancer development.

Acquired factor VIII inhibitor in a non-haemophilic boy.

We describe the case of a previously healthy 8-year-old non-haemophilic boy who developed a factor VIII inhibitor of unknown origin. The symptoms of this disease were haemorrhages in the muscles of the right thigh, numerous bruises and a large haematoma of the right crus with subsequent tissue necrosis. Activated and non-activated prothrombin complex concentrates were administered in the therapy of the haemorrhages. To eliminate factor VIII inhibitor, the patient was treated first with prednisone, then immunoglobulin G and finally with a combination of prednisone and cycylophosphamide, without any effect. A total spontaneous remission was observed after 15 months from the beginning of the disease.

[Molecular diagnosis of lymphomas and leukaemias]. / Diagnostyka molekularna chloniaków I bialaczek.

In the last years molecular diagnosis has become a routine method m the evaluation of haematological malignancies. Various techniques are used to assess B- and T-cell clonality, chromosomal rearrangements involving protooncogenes, monitoring of minimal residual disease as well as genome wide scanning methods such as comparative genomic hybridization or spectral karyotyping. The review explores some avenues in which molecular techniques can affect patient's retrospective and prospective diagnosis. The major advantages and disadvantages of some of these techniques are described and their place in the scheme of diagnosis and treatment is briefly summarised.

Expression of CD20 on acute lymphoblastic leukemia cells in children.

CD20 determinant expressed on B precursors is associated with regulation of proliferation, apoptosis and maturation of these cells. The acute lymphoblastic leukemia "common" type (cALL) based on expression of CD20 is subdivided in type I and II. However, the clinical significance of CD20 expression on cALL and significance of cALL type I and II discernment are not fully elucidated. The association of CD20 expression with the expression of multidrug resistance molecule (MDR), CD34, atypical immunophenotypes of leukemia cells and response to induction therapy were determined in the group of 147 patients with acute lymphoblastic leukemia (ALL) B progenitor type (ALL-proB -14 patients) and common type (cALL-133 patients). The expression of CD20 on leukemia cells was studied routinely at diagnosis before the therapy. This expression was noted on leukemia cells of 6 ALL-proB patients (42.8%) and 66 cALL patients (49.6%). The expression of CD20 showed no association with the expression of CD34, CD22 and MDR. The reverse association was observed between CD20 expression and the presence of co-expression of myeloid (CD13, CD33, CD65, CD15) and T lymphoid determinants (CD2, CD5, CD7) on leukemia cells. The effect of induction therapy analyzed as time of blast cells cytoreduction in peripheral blood and time of reaching the complete remission showed the slower clearance of peripheral blood from blast cells associated with expression of CD20. There was no association of CD20 expression with the time of reaching the hematological remission. The above results suggested a "protective" role of CD20 against co-expression of other determinants (myeloid and lymphoid) and no association with the results of induction therapy.

Release of cytokines and soluble cytokine receptors after intravenous anti-D treatment in children with chronic thrombocytopenic purpura.

INTRODUCTION: Immunoglobulin anti-D administration is one of several methods used in treating children with chronic immune thrombocytopenic purpura. Fc receptor blockade of the reticuloendothelial cell system and of mononuclear phagocytes is an important mechanism of the action of anti-D in ITP. Recently other possible mechanisms by which anti-D works in ITP have been considered. METHODS: The aim of this study was to obtain a better understanding of the effect of anti-D administration on cytokine, soluble cytokine receptors and platelet count in children with chronic ITP and to determine the pre-treatment plasma cytokine profile in this group of patients. Eighteen children with chronic ITP were examined. In our study the impact of anti-D on the cytokine network was evaluated by analysing serum levels of IL-6, IL-8, tumor necrosis alpha and soluble TNF receptors I and II by the EASIA method before and 1, 3, 20 and 40 h then seven days and one month after anti-D infusion. RESULTS: Anti-D caused a significant increase in platelet count 20 h postinfusion in 10 out of 18 children, 96 h postinfusion in three children and 168 h postinfusion in one child. The mean duration of the response was four weeks. A significant and rapid increase in plasma levels of IL-6, IL-8 and TNF-alpha was seen within 1 to 20 h after anti-D infusion. This increase was accompanied by a prolonged elevation of soluble TNF receptors. There was a significant correlation between TNF-alpha and IL-8, IL-8 and IL-6, TNF-alpha and sTNFRI, and sTNF receptors I and II. CONCLUSION: These data demonstrate that anti-D infusion caused changes in the cytokine network and raises the question of whether the therapeutic effectiveness of anti-D is related to its immunomodulating properties.

[Use of intratympanic gentamycin for the treatment of Meniere's disease]. / Transtympanalne podawanie gentamycyny w chorobie Meniere'a.

The ablation treatment of Meniere's disease by intratympanic streptomycin applications was first reported by Schuknecht in 1957. Streptomycin and gentamycin are the most frequent aminoglycosides used for the Meniere's disease treatment. Gentamycin is responsible for the damage of vestibular dark cells causing the impairment of endolymph production. This method gives the possibility for the control of the vertigo with a potential hearing preservation. Fifteen patients with unilateral Meniere's disease who had not responded to conventional therapy, were treated by intratympanic gentamycin injections. The hearing status and the caloric test were staged before and after treatment according to the AAO-HNS guidelines. Overall results after minimum 1 year follow-up in this group were as shown below: complete vertigo control--5 patients, substantial vertigo control--8 patients, complete relief of tinnitus--10, relief of aural fullness--12 patients, hearing loss--none. According to presented results, intratympanic injections of gentamycin is the useful alternative to the surgery. This method should be consider in every patient with the unilateral Meniere's disease, who had not responded to the conventional treatment.

[The great investigators of the ear anatomy]. / Wielcy badacze anatomii ucha.

There was presented the galaxy of famous anatomists which had investigated the structure of the ear and the temporal bone. There were shown the anatomical parts of the ear, which were named from the names of their discoverers.

[Results of the treatment of myelodysplastic syndrome (MDS)obtained by the Polish Paediatric Leukaemia /Lymphoma Study Group]. / Wyniki leczenia zespolów mielodysplastycznych (MDS) uzyskane przez Polska Pediatryczna Grupe ds Leczenia Bialaczek i Chloniaków u dzieci.

Sixty children with MDS treated in six centres of the Polish Paediatric Leukaemia/Lymphoma Study Group in the period 1975-1999 were included in the study. In 20 children RAEB-T, in 13 RA, in 21 RAEB and in 6 CMML were diagnosed. Our own and literature data showed that BMT is the best therapy for children with MDS. We need a new comprehensive protocol for the diagnosis and treatment of children with MDS in Poland.

Determination of flumequine and doxycycline in milk by a simple thin-layer chromatographic method.

Tetracycline and quinolone antibiotics have for many years served as important classes of veterinary drugs. Two representatives of both classes: doxycycline from tetracyclines and flumequine from quinolones are often administered together. When the withdrawal periods are not obeyed, the antibiotic residues may be present in edible products, e.g., in meat, eggs or milk. In the present paper a simple thin-layer chromatography (TLC) screening method is established for determining these drugs in milk. Only two developments of the plate with concentrating zone are needed: one as a clean-up procedure, the other as a proper analysis. The spots were detected both by UV lamp with dual wavelength (254 and 366 nm) and by densitometry.

[Melkersson-Rosenthal syndrome]. / Zespól Melkerssona-Rosenthala.

The Melkersson-Rosenthal syndrome consists of a triad of symptoms: peripheral facial nerve paralysis, congenital "lingua plicata" and noninflammatory facial oedema. The authors present 2 cases with complete picture of this syndrome and some review of the literature.