Jana Chovancová

Ofatumumab added to dexamethasone in patients with relapsed or refractory chronic lymphocytic leukemia: Results from a phase II study

The treatment of relapsed/refractory chronic lymphocytic leukemia (CLL) remains a challenging clinical issue. An important treatment option is the use of high‐dose corticosteroids. The purpose of this clinical trial was to determine the efficacy and toxicity of an ofatumumab–dexamethasone (O‐Dex) combination in relapsed or refractory CLL. The trial was an open‐label, multicenter, nonrandomized, Phase II study. The O‐Dex regimen consisted of intravenous ofatumumab (Cycle 1: 300 mg on day 1, 2,000 mg on days 8, 15, and 22; Cycles 2–6: 1,000 mg on days 1, 8, 15, and 22) and oral dexamethasone (40 mg on days 1–4 and 15–18; Cycles 1–6). The O‐Dex regimen was given until best response, or a maximum of six cycles. Thirty‐three patients (pts) were recruited. Twenty‐four (73%) pts completed at least three cycles of therapy. The remaining nine pts were prematurely discontinued owing to Grade 3/4 infections (seven pts), disease progression (one pt), or uncontrollable diabetes mellitus (one pt). Overall response rates/complete remissions (ORR/CR) were achieved in 22/5 pts (67/15%). The median progression‐free survival (PFS) was 10 months. In pts with p53 defects (n = 8), ORR/CR were achieved in 5/2 pts (63/25%) with a median PFS of 10.5 months. The median overall survival (OS) was 34 months. The Grades 3–5 infectious toxicity in 33% of pts represented the most frequent side effect during the treatment period. In conclusion, the O‐Dex regimen shows a relatively high ORR and CR with promising findings for PFS and OS. The study was registered at www.clinicaltrials.gov (NCT01310101). Am. J. Hematol. 90:417–421, 2015.

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.

Detecting minimal residual disease in patients with chronic lymphocytic leukemia using 8-color flow cytometry protocol in routine hematological practice.

Minimal residual disease (MRD) detection has become increasingly important for the assessment of therapy response in chronic lymphocytic leukemia (CLL). However, current MRD analysis methods, both molecular genetic and flow cytometric, are time‐consuming and require experienced laboratory staff.

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.

Detection of minimal residual disease in mantle cell lymphoma—establishment of novel eight-color flow cytometry approach

Minimal residual disease (MRD) detection is an essential tool for therapy response assessment in a considerable number of hematooncologic disorders, including mantle cell lymphoma (MCL). Flow cytometry (FCM) ranks among the most effective approaches, which allows rapid sample processing and compete successfully with highly sensitive molecular methods like polymerase chain reaction. Because FCM is ordinarily applied to detect MRD in B‐lineage diseases like chronic lymphocytic leukemia, a similar method could be used in MCL. We decided to test our novel eight‐color FCM approach in MCL MRD detection.

Cytokine analysis in a patient with relapsing Kikuchi - Fujimoto disease

In 2006, a 26-year-old woman presented with tender cervical and retroauricular lymphadenopathy accompanied by fever, lower limb paraparesis, skin rash and generalized myalgia. Her past medical history was unremarkable. Full blood count and renal and hepatic profi les were within normal ranges, with the exception of mild lactate dehydrogenase elevation (237 U/L). Microbiological and radiological screening revealed no infectious etiology, and immunological fi ndings were negative or normal. In contrast to increased C-reactive protein levels (140 mg/L), the serum procalcitonin level was low. Brain magnetic resonance and lumbar puncture showed no pathology.

Ability to downregulate the level of cyclin-dependent kinase inhibitor p27(Kip1) after DNA damage is retained in chronic lymphocytic leukemia cells with functional ATM/p53 signaling pathway.

The activity of the key DNA-damage response (DDR) kinases, ATR and ATM, is largely compromised in chronic lymphocytic leukemia (CLL) cells. While noncycling CLL cells lack ATR protein expression,[1] ATM is commonly targeted for inactivation by genetic abnormalities of the ATM gene.[2] It has been shown that checkpoint pathways, which operate in cycling somatic cells and target Cdk2 kinase activity, are to some extent activated in arrested CLL cells after DNA damage as well;[3] however, the impact of these pathways is questionable, as it could be hampered by predominantly cytoplasmic localization of Cdk2 in CLL cells.[4] Nevertheless, DDR plays a key role in sensitivity of CLL to chemotherapy. Another stress-response kinase, p38 MAP kinase (p38MAPK), was shown to promote CLL cells survival.[5] In multiple cell types, p38MAPK activates different cell cycle checkpoints (mainly through regulation of cyclindependent kinase (Cdk) inhibitors p21Cip1 and p27Kip1), and also ATM/ATR-dependent G2/M checkpoint signaling through MAPKAP kinase-2 activity.[6] While relationship between ATM/p53 and p21Cip1 expression in CLL was studied,[7] the association of ATM kinase activity with the levels of p27Kip1 after DNA damage has not been elucidated in CLL cells.

NOD/SCID IL2Rγ-null mouse xenograft model of human p53-mutated chronic lymphocytic leukemia and ATM-mutated mantle cell lymphoma using permanent cell lines.

Xenograft models represent a promising tool to study the pathogenesis of hematological malignancies. To establish a reliable and appropriate in vivo model of aggressive human B-cell leukemia and lymphoma we xenotransplanted four p53-mutated cell lines and one ATM-mutated cell line into immunodeficient NOD/SCID IL2Rγ-null mice. The cell lines MEC-1, SU-DHL-4, JEKO-1, REC-1, and GRANTA-519 were transplanted intraperitoneally or subcutaneously and the engraftment was investigated using immunohistochemistry and flow cytometry. We found significant differences in engraftment efficiency. MEC-1, JEKO-1 and GRANTA-519 cell lines engrafted most efficiently, while SU-DHL-4 cells did not engraft at all. MEC-1 and GRANTA-519 massively infiltrated organs and the whole intraperitoneal cavity showing very aggressive growth. In addition, GRANTA-519 cells massively migrated to the bone marrow regardless of the transplantation route. The MEC-1 and GRANTA-519 cells can be especially recommended for in vivo study of p53-mutated chronic lymphocytic leukemia and ATM-mutated mantle cell lymphoma, respectively.

The influence of mutational status and biological characteristics of acute myeloid leukemia on xenotransplantation outcomes in NOD SCID gamma mice

PurposeThis study aimed at analyzing the association of gene mutations and other acute myeloid leukemia (AML) characteristics with engraftment outcomes in immunodeficient mice and to select the engraftment outcomes that best reflect patient survival.MethodsMutations in 19 genes as well as leukemia- and patient-related characteristics were analyzed for a group of 47 de novo AML samples with respect to three engraftment outcomes: engraftment ability, engraftment intensity (percentage of hCD45+ cells) and engraftment latency. Leukemia-related characteristics were additionally analyzed in an extended group of 68 samples that included the 47 de novo samples, and additional 21 samples from refractory and relapsed cases. Engraftment outcomes were compared with overall and event-free survival of the patients.ResultsFor the 47 de novo samples, no single mutation influenced engraftment, whereas the NPM1mut/DNMT3Amut co-mutation was associated with higher engraftment ability. NPM1mut/FLT3-ITDneg had lower engraftment intensity. Among leukemia-related characteristics, a complex karyotype was associated with higher engraftment intensity. Among patient-related characteristics, higher cytogenetic risk was associated with higher engraftment intensity, and failure to achieve clinical remission was associated with shorter engraftment latency. In the extended group of 68 samples, white blood count was associated with higher engraftment ability, and the presence of a complex karyotype was associated with higher engraftment intensity. Association with patient overall survival was seen only for engraftment intensity.ConclusionsThe engraftment of AML was influenced by mutation-interactions and other AML characteristics, rather than by single mutated genes, and engraftment intensity best reflected clinical penetrance of AML.

Local chemical sympathectomy of rat bone marrow and its effect on marrow cell composition

Existing experimental studies of the effect of sympathetic nerve fibers on bone marrow cells are based on the systemic administration of neurotoxic 6-hydroxydopamine. The method of global chemical sympathectomy has some serious disadvantages and could lead to questionable results. We describe a new method of local chemical sympathectomy of rat femoral bone marrow using guanethidine (Ismelin) delivery using an osmotic mini pump. Local guanethidine treatment for 14days led to complete elimination of sympathetic fibers in femoral bone marrow in contrast to bone marrow of contralateral or naïve femurs. Ablation of sympathetic fibers was associated with a loss of rat endothelial cell marker (RECA) indicating immunophenotype changes in blood vessel endothelial cells, but no significant effect of guanethidine was found on the survival of endothelial cells and mesenchymal stem cells in vitro. Moreover, local guanethidine treatment also elicited a significant reduction of Nestin+/SDF1+ mesenchymal stem cells and c-Kit+/CD90+ hematopoietic stem cells in femoral bone marrow. Tissue-specific chemical sympathectomy of rat bone marrow by guanethidine overcomes some of the drawbacks of systemic administration of neurotoxic compounds like 6-hydroxydopamine and delivers unequivocal evidence on the effects of sympathetic innervation on the cell content of bone marrow.