Martin Klabusay

Monoallelic and biallelic inactivation of TP53 gene in chronic lymphocytic leukemia: selection, impact on survival, and response to DNA damage

Deletion of TP53 gene, under routine assessment by fluorescence in situ hybridization analysis, connects with the worst prognosis in chronic lymphocytic leukemia (CLL). The presence of isolated TP53 mutation (without deletion) is associated with reduced survival in CLL patients. It is unclear how these abnormalities are selected and what their mutual proportion is. We used methodologies with similar sensitivity for the detection of deletions (interphase fluorescence in situ hybridization) and mutations (yeast functional analysis) and analyzed a large consecutive series of 400 CLL patients; a subset of p53-wild-type cases (n = 132) was screened repeatedly during disease course. The most common type of TP53 inactivation, ie, mutation accompanied by deletion of the remaining allele, occurred in 42 patients (10.5%). Among additional defects, the frequency of the isolated TP53 mutation (n = 20; 5%) and the combination of 2 or more mutations on separate alleles (n = 5; 1.3%) greatly exceeded the sole deletion (n = 3; 0.8%). Twelve patients manifested defects during repeated investigation; in all circumstances the defects involved mutation and occurred after therapy. Monoallelic defects had a negative impact on survival and impaired in vitro response to fludarabine. Mutation analysis of the TP53 should be performed before each treatment initiation because novel defects may be selected by previous therapies.

Cell Therapy in Patients with Left Ventricular Dysfunction Due to Myocardial Infarction

Objectives: The purpose of this study was to determine the impact of autologous transplantation of mononuclear bone marrow cells on myocardial function in patients with left ventricular (LV) dysfunction due to an acute myocardial infarction. Methods: The randomized study included 82 patients with a first acute myocardial infarction treated with a stent implantation. This presentation is a subanalysis of 47 patients with left ventricular dysfunction–EF (ejection fraction) ≤ 40%. Group H patients (n = 17) received higher number (100,000,000) of cells; Group L patients (n = 13) received lower number (10,000,000) of cells. The patients of control Group C (n = 17) were not treated with cells. The Doppler tissue imaging and single photon emission computed tomography were performed before cell transplantation and 3 months later. Results: At 3 months of follow‐up, the baseline EF of 35%, 36%, 35% in Groups H, L, and C increased by 6% (P < 0.01 vs. baseline), 5% (P < 0.01 vs. baseline), and 4% (P = NS vs. baseline), respectively, as assessed by single photon emission computed tomography (P = NS between groups). The baseline number of akinetic segments of 6.9, 7.0, and 6.2 in H, L, and C groups decreased by 1.7 (P < 0.01 vs. baseline), 1.5 (P < 0.01 vs. baseline), and 0.7 (P = NS vs. baseline, P = NS between groups), respectively, as demonstrated by echocardiography. Conclusion: In our study, the statistically important effect of transplantation of mononuclear bone marrow cells on myocardial function was not found. Only an insignificant trend toward the improvement of global LV EF fraction was found at 3‐month follow‐up.

Different levels of CD52 antigen expression evaluated by quantitative fluorescence cytometry are detected on B‐lymphocytes, CD 34+ cells and tumor cells of patients with chronic B‐cell lymphoproliferative diseases

The success of treatment using monoclonal antibodies in oncology is influenced by, among other factors, the level of target antigen expression on tumor cells. The authors analyzed the intensity of the CD52 antigen expression in patients with chronic lymphoproliferative diseases and compared them with B‐lymphocytes of a healthy population and CD34+ cells in peripheral blood stem cells (PBSC) grafts.

Granulocyte maturation determines ability to release chromatin NETs and loss of DNA damage response; these properties are absent in immature AML granulocytes.

Terminally-differentiated cells cease to proliferate and acquire specific sets of expressed genes and functions distinguishing them from less differentiated and cancer cells. Mature granulocytes show lobular structure of cell nuclei with highly condensed chromatin in which HP1 proteins are replaced by MNEI. These structural features of chromatin correspond to low level of gene expression and the loss of some important functions as DNA damage repair, shown in this work and, on the other hand, acquisition of a new specific function consisting in the release of chromatin extracellular traps in response to infection by pathogenic microbes. Granulocytic differentiation is incomplete in myeloid leukemia and is manifested by persistence of lower levels of HP1γ and HP1β isoforms. This immaturity is accompanied by acquisition of DDR capacity allowing to these incompletely differentiated multi-lobed neutrophils of AML patients to respond to induction of DSB by γ-irradiation. Immature granulocytes persist frequently in blood of treated AML patients in remission. These granulocytes contrary to mature ones do not release chromatin for NETs after activation with phorbol myristate-12 acetate-13 and do not exert the neutrophil function in immune defence. We suggest therefore the detection of HP1 expression in granulocytes of AML patients as a very sensitive indicator of their maturation and functionality after the treatment. Our results show that the changes in chromatin structure underlie a major transition in functioning of the genome in immature granulocytes. They show further that leukemia stem cells can differentiate ex vivo to mature granulocytes despite carrying the translocation BCR/ABL.

Presence of heterozygous ATM deletion may not be critical in the primary response of chronic lymphocytic leukemia cells to fludarabine.

Objectives:  Abnormalities of the TP53 or ATM, cooperating tumor‐suppressor genes, significantly worsen the treatment options for chronic lymphocytic leukemia (CLL) patients. Although the aberrations seem to be mutually exclusive in this leukemia, inactivation of the former gene leads to worse prognosis. We tested the in vitro sensitivity of the CLL samples with heterozygous ATM deletion to fludarabine and combination of fludarabine and rituximab; the responses were compared with the TP53‐abnormal and wild‐type (wt) cells to delimitate relative significance of ATM deletion.

Lenalidomide proved effective in multisystem Langerhans cell histiocytosis

Langerhans cell histiocytosis (LCH) is a rare idiopathic disease with diverse clinical manifestations ranging from a single osteolytic lesion to generalized disease. Various treatment regimens have been proposed for the multisystem type, however, with inconsistent outcomes. Herein we are the first to report on a therapy effect of a lenalidomide-based regimen in a patient with repeatedly relapsed aggressive form of multisystem LCH.

Intracoronary Delivery of Bone Marrow Cells to the Acutely Infarcted Myocardium

Objectives: Intracoronary cell transplantation during catheter balloon inflations may be associated with adverse events. We studied the effectiveness of an alternative transplantation technique – intracoronary cell infusion. Methods: Fourteen pigs, which had survived acute myocardial infarction, were randomized into 2 treatment groups and 2 controls. Three days after infarction, 12 pigs underwent allogeneic intracoronary mononuclear bone marrow cell transplantation using either the standard technique (short-term cell injections during repeat balloon inflations, technique A, n = 6) or continuous intracoronary cell infusion without balloon inflations (technique B, n = 6). Implanted cells were stained with fluorescent dye. After transplantation, the pigs were euthanized and myocardial samples were analyzed by fluorescent microscopy. Results: The mean numbers of fluorescently labeled bone marrow cells in the infarction border zone, in the infarction mid-area and in the center of myocardial infarction were 84, 72 and 55 using technique A, and 29, 57 and 46 using technique B, respectively. The mean cell retention in the infarction border zone of 84 cells for technique A and 29 cells for technique B differed significantly (p = 0.034, two-tailed t test). Conclusion: The continuous intracoronary cell infusion technique is a less efficient cell delivery technique as compared with the standard technique using repeat intracoronary balloon inflations.

Lenalidomide: a new treatment option for Castleman disease

A male born in 1961, aged 46, was referred to our department for evaluation of splenomegaly and generalized lymphadenopathy aff ecting the cervical, axillary, mediastinal, retroperitoneal and inguinal regions. Laboratory data revealed an increased erythrocyte sedimentation rate (16 mm/h and 30 mm/2 h) and C-reactive protein level (35.4 mg/L). Total protein and full blood counts as well as renal and hepatic profi les were within normal ranges, and microbiological screening revealed no infectious etiology. Other fi ndings, including radiological examinations and bone marrow biopsy, showed no further pathologies. Th e patient ’ s other medical history was signifi cant for arterial hypertension, chronic infl ammatory demyelinating polyneuropathy and mild right hemi-paresis with expressive language disorder. Based on lymph node biopsies from retroperitoneal and both inguinal regions, the patient was diagnosed with the plasma-cell variant of CD. Th e presence of a large pelvic mass compressing adjacent structures indicated the patient for therapy initiation. During fi rst-line treatment (R-CHOP: rituximab 375 mg/m 2 , cyclophosphamide 750 mg/m 2 , doxorubicin 50 mg/m 2 and vincristine 2 mg intravenously on day 1; prednisone 100 mg perorally on days 1 – 5 in a 21-day cycle, three cycles in total, 12/2008 – 2/2009), clinical progression of the disease was evident (gastrointestinal symptoms, swollen hands and feet with signs of vasculitis), and there was no radiological response on a restaging computed tomography (CT) examination after 3 months.

Retention of nanoparticles-labeled bone marrow mononuclear cells in the isolated ex vivo perfused heart after myocardial infarction in animal model.

Cell therapy of myocardial infarction (MI) is under clinical investigation, yet little is known about its underlying mechanism of function. Our aims were to induce a sub-lethal myocardial infarction in a rabbit, to evaluate the abilities of labeled bone marrow mononuclear cells to migrate from the vessel bed into extracellular space of the myocardium, and to evaluate the short-term distribution of cells in the damaged left ventricle. Sub-lethal myocardial infarction was induced in rabbits by ligation of the left coronary vessel branch (in vivo). The Langendorff heart perfusion model (ex vivo) was used in the next phase. The hearts subjected to MI induction were divided into 3 groups (P1–P3), and hearts without MI formed a control group (C). Nanoparticles-labeled bone marrow mononuclear cells were injected into coronary arteries via the aorta. Perfusion after application lasted 2 minutes in the P1 group, 10 minutes in the P2 and C groups, and 25 minutes in the P3 group. The myocardium of the left ventricle was examined histologically, and the numbers of labeled cells in vessels, myocardium, and combined were determined. The numbers of detected cells in the P1 and C groups were significantly lower than in the P2 and P3 groups. In the P2 and P3 groups, the numbers of cells found distally from the ligation were significantly higher than proximally from the ligation site. Bone marrow mononuclear cells labeled with iron oxide nanoparticles proved the ability to migrate in the myocardium interstitium with significantly higher affinity for the tissue damaged by infarction.

Magnetizable Duplex Steel Stents Enable Endothelial Cell Capture

Emerging medical nanotechnology applications often utilize magnetic forces to guide the movement of superparamagnetic particle linked cells and drugs in order to achieve a therapeutic effect. Superparamagnetic particle labeled endothelial cells have previously been captured on the surface of prototype nickel-plated stents in proof of concept studies. Facilitated endothelialization may help improve the healing of stented arteries and reduce the risk of stent thrombosis and restenosis. Extensive evaluation of candidate materials led to the development of a magnetizable 2205 duplex stainless steel stent. Magnetic field strengths of approximately 630 mG were induced within these stents by holding them in close proximity to a 0.7 T rare earth magnet. The magnetic field strength was reliably maintained over several days, but was partially reduced upon mild mechanical shock or plastic deformation. Mechanical testing demonstrated that stents could withstand crimping and expansion necessary for vascular implantation; however, magnetic field strength was significantly reduced. When placed in an endothelial cell suspension of 1×106 cells/mL, magnetized stents captured approximately 310 cells/mm2 compared to approximately 35 cells/mm2 for non-magnetized control stents. These data provide quantitative support to the observation that low level magnetization of stents may be adequate to attract labeled, autologous, blood-derived endothelial outgrowth cells following stent placement. This, in turn, may lead to more rapid and complete healing of stented arteries with a concomitant improvement in stent performance.