Alexey V. Danilov

Molecular Pathogenesis of Chronic Lymphocytic Leukemia

Chronic lymphocytic leukemia (CLL) is unique among malignancies since it represents an accumulation of B-lymphocytes resistant to apoptosis. Several factors are thought to confer this unusual feature to a CLL B-cell. Misbalance between cytoplasmic pro-survival and pro-death molecules, such as Bcl-2, Mcl-1 and alike, appears to be one of the key factors defining B-cell longevity. Autocrine pathways, such as vascular endothelial growth factor-receptor pathway, also contribute to survival. The role of B-cell receptor (BCR) is less straightforward. In the last decade it became clear that CLL does not constitute a uniform disease, but, based on the prevalence of mutations in the BCR heavy chain (IgVH), can be classified into two distinct subgroups. Several molecular markers correlate with IgVH mutations. Some of them, like zeta-chain associated protein kinase, are also involved in BCR signaling and influence cell cycle. Yet the primary pathogenic event leading to increased proliferation and survival in CLL is difficult to ascertain. Molecules involved in BCR signaling pathways and cytoplasmic pro-survival players probably act in concert to confer resistance to apoptosis. In this respect, the role of the B-CLL environment, which includes nurse-like cells and T-cells, cannot be underestimated. Nurse-like cells provide stimuli necessary for perpetuation of life in CLL. On the other hand, abnormal T-cell function, whether it is excessive immunosuppression delivered by regulatory T-cells or insufficient anti-tumor immunity rendered by T-helpers, allows malignant CLL cells to go unnoticed by the cellular immune system.

Managing a pregnant patient with paroxysmal nocturnal hemoglobinuria in the era of eculizumab

Paroxysmal nocturnal hemoglobinuria (PNH) is a rare clonal stem cell disorder, which affects women of child-bearing age. PNH is associated with thrombotic complications, which are the main causes of morbidity and mortality. Management of a pregnant woman with PNH remains a challenge due to high incidence of thrombotic complications and the difficulty of differentiating a PNH crisis from the complications of pregnancy. PNH is associated with an increased rate of premature labor and fetal loss. Eculizumab, a humanized monoclonal antibody directed against the terminal complement protein C5, has revolutionized treatment of PNH. However, the role of eculizumab in pregnancy is unclear. We review the current strategies for the management of pregnant women with PNH, underline the controversies and present our recommendations.

Decitabine is an effective treatment of idiopathic myelofibrosis.

Idiopathic myelofibrosis (IMF) is a progressive chronic myeloproliferative disorder. The median survival for patients with IMF is 70 months with limited treatment options (Tefferi et al, 2007a). At this time, allogeneic stem cell transplantation is the only curative treatment modality in IMF. However, it is accompanied by high transplant-related mortality. A number of potential treatment regimens have been proposed but their clinical efficacy remains suboptimal (Cervantes et al, 2007). Epigenetic modifications have been implicated in the pathogenesis of IMF, resulting in rapid cell proliferation and circulating abnormal progenitors with extramedullary haematopoiesis (Wang et al, 2002; Jones et al, 2004; Bogani et al, 2008). This suggests that hypomethylating agents can be of therapeutic value in IMF. Azacitidine had a modest efficacy in a recent Phase II trial, documenting a clinical improvement in 21% of patients (Quintas-Cardama et al, 2008). Here we report on the successful use of the hypomethylating agent Decitabine (Dacogen, SuperGen, Inc., Dublin, CA, USA) in the treatment of an elderly patient with symptomatic transfusiondependent IMF. A 65-year-old white male with history of atrial fibrillation presented in March 2004 with worsening fatigue and generalized pruritus. On physical examination, he had prominent splenomegaly and hepatomegaly. The spleen was palpable 4–6 cm below the left costal margin. The complete blood count showed: white blood cell (WBC) count, 8Æ0 · 10/l with normal differential; haemoglobin concentration (Hb), 83 g/l; haematocrit 26Æ8% with mean cell volume 109 fl; platelet count 565 · 10/l. Indirect bilirubin was elevated at 37Æ6 lmol/l, lactate dehydrogenase was 217 IU/l. A peripheral blood smear demonstrated increased numbers of target cells, numerous teardrop forms, large platelets and rare nucleated red blood cells. The bone marrow biopsy was a markedly hypercellular marrow with increased and atypical megakaryocytes, with extensive fibrosis consistent with cellular phase of IMF (Tefferi et al, 2007b). Cytogenetic studies on the bone marrow and blood were negative for t(9;22) and the JAK2 V617F mutation. The patient received a brief trial of darbepoietin for treatment of his symptomatic anemia. He responded but continued to require packed red blood cell (PRBC) transfusions, averaging two units per month, for symptomatic anaemia. Pruritus was successfully managed with low doses of prednisone. Because of the patient’s progressive symptomatic splenomegaly and thrombocytosis he was started on hydroxycarbamide. In an attempt to decrease the PRBC transfusion requirements lenalinomide was initiated at a low dose of 10 mg/d. However, the patient’s transfusion requirement increased further to 8–16 units of PRBC per month. In September 2007, he complained of increasing abdominal fullness with early satiety and weight loss. His WBC count was 4Æ9 · 10/l, Hb 75 g/l, haematocrit 22Æ8%, and platelet count 133 · 10/l. The peripheral blood smear demonstrated marked anisocytosis with macrocytes, abundant teardrop forms, nucleated red blood cells and abnormally lobulated and hypogranular neutrophils with cytoplasmic vacuoles, giant platelets and rare myelocytes and promyelocytes. A repeat bone marrow biopsy again revealed a hypercellular marrow with dysplastic myeloid and erythroid precursors. Megakaryocytes were atypical and increased in number and there was a diffuse increase in reticulofibrosis (3/ 4). Cytogenetic studies were normal. Decitabine was started at a dose of 20 mg/m for 5 d every 4 weeks. Following two cycles of decitabine, the patient’s transfusion requirement decreased to two units of PRBC per month. He gained weight and reported a substantial increase in his energy level. His spleen decreased from 10–12 cm to 2 cm below the left costal margin. He has continued on decitabine. His transfusion requirement decreased further. After six cycles, the WBC count remained 2Æ6 · 10/l, Hb 89 g/l, haematocrit 26Æ9 vol % and platelet count 274 · 10/l. He has tolerated decitabine well with a performance status of 90%. In vitro studies demonstrate that hypomethylating agents can reduce the number of circulating malignant progenitor cells in IMF (Shi et al, 2007). Treatment of peripheral blood CD34 cells from patients with IMF with decitabine followed by a histone deacetylase inhibitor, trichostatin A, result in upregulation of CXCR4 expression restoring their ability to migrate in response to stromal-derived factor-1a (Shi et al, 2007). Decitabine decreased the transfusion requirement, splenomegaly and resulted in a marked clinical improvement in our symptomatic transfusion-dependent patient with IMF. Larger studies are warranted to evaluate the efficacy of decitabine in patients with IMF.

Differential control of G0 programme in chronic lymphocytic leukaemia: a novel prognostic factor

Chronic lymphocytic leukaemia (CLL) is a unique malignancy where quiescent B cells accumulate in the peripheral blood. Since clinical outcomes in CLL are very heterogeneous, it is of utmost importance to correctly assess the disease prognosis in each individual case. Recently, it has been shown that high ZAP‐70 [Zeta‐chain (T‐cell receptor) associated protein kinase (70 kDa)] expression level strongly correlates with lack of IgVH mutations and poor prognosis in B‐CLL. As CLL malignant cells are arrested in G0, we investigated whether Dipeptidyl Peptidase 2 (DPP2), a serine protease that plays a key role in keeping cells in the quiescent state, is involved in cell‐cycle control in CLL. We have previously shown that specific inhibition of DPP2 results in apoptosis of normal lymphocytes. In this study, cell apoptosis experiments were conducted in 38 patients with B‐CLL. Two distinct subsets of B‐CLL were identified, susceptible and resistant to DPP2‐inhibition‐induced apoptosis. If resistant to apoptosis (42·1%), the CLL cells have higher expression of ZAP‐70 and exhibit a worse prognosis, such as shorter treatment‐free time period. Thus, resistance vs. susceptibility to DPP2‐inhibiton induced apoptosis can be employed as a novel prognostic factor in CLL.

Dipeptidyl Peptidase 2 apoptosis assay determines the B-cell activation stage and predicts prognosis in chronic lymphocytic leukemia

OBJECTIVE Dipeptidyl peptidase 2 (DPP2/DPP7) is a regulator of quiescence as inhibition of DPP2 results in apoptosis of resting, but not activated lymphocytes. The purpose of the present study was to investigate the prognostic value of DPP2 inhibition and the role of DPP2 in cell cycle in chronic lymphocytic leukemia (CLL). MATERIALS AND METHODS We screened 152 peripheral blood samples from patients with CLL in an apoptosis assay with AX8819, a DPP2-specific inhibitor. The apoptotic response was correlated with B-cell receptor signaling and cell cycle and molecular prognostic factors. RESULTS We categorized CLL into two prognostic subgroups. Inhibition of DPP2 induced apoptosis in 60% of CLL, while 40% were resistant to apoptosis. Resistance to apoptosis correlated with unmutated IgV(H) and increased ZAP-70 expression and was associated with unfavorable clinical outcomes. Sensitive CLL B cells expressed high p27, low c-Myc protein levels and decreased Syk phosphorylation, indicative of a resting phenotype. DPP2 inhibition in those cells resulted in apoptosis accompanied by enhanced phosphorylation of Syk, degradation of p27 and p130, and upregulation of c-Myc, indicative of activation and inappropriate cell cycle entry. Resistant CLL demonstrated baseline low p27 and high c-Myc protein levels and increased pSyk, indicative of an activated phenotype. Inhibition of heat shock protein 90 in this subset of CLL partially reversed apoptosis resistance. CONCLUSIONS The DPP2 apoptosis assay provides a reliable prognostic factor in CLL. CLL B cells sensitive to DPP2 inhibition are in true G(0), while resistant CLL B-cells are partially activated. DPP2 inhibition alone or with concomitant inhibition of heat shock protein 90 warrants investigation as a therapeutic modality in CLL.

Cell cycle control and adhesion signaling pathways in the development of metastatic melanoma

Metastatic melanoma is a fatal malignancy which is remarkably resistant to treatment. It is not entirely clear what determines transition from primary local to metastatic melanoma. Recent gene profiling studies shed light onto the complexity of pathogenesis of melanoma progression. An interaction between cell cycle signaling, adhesion pathways and epithelial–mesenchimal transition program appears to be critical in the development of metastatic disease. An isolated deregulation of either of those pathways may not be sufficient to initiate tumor evolution towards an aggressive phenotype. Here we review how they act in concert to make such a transition possible.