Double-strand breaks (DSBs) are the most deleterious DNA lesions, which, if left unrepaired, may have severe consequences for cell survival, as they lead to chromosome aberrations, genomic instability, or cell death. Various physical, chemical, and biological factors are involved in DSB induction. Cells respond to DNA damage by activating the so-called DNA damage response (DDR), a complex molecular mechanism developed to detect and repair DNA damage. The formation of DSBs triggers activation of many factors, including phosphorylation of the histone variant H2AX, producing γH2AX. Phosphorylation of H2AX plays a key role in DDR and is required for the assembly of DNA repair proteins at the sites containing damaged chromatin as well as for activation of checkpoints proteins which arrest the cell cycle progression. In general, analysis of γH2AX expression can be used to detect the genotoxic effect of different toxic substances. When applied to clinical samples from cancer patients, evaluation of γH2AX levels may allow not only to monitor the efficiency of anticancer treatment but also to predict of tumor cell sensitivity to DNA damaging anticancer agents and toxicity of anticancer treatment toward normal cells.

The generally accepted mechanism of metformin’s effect is stimulation of adenosine monophosphate (AMP)-activated protein kinase (AMPK). AMPK is directly activated by an increase in AMP:ATP ratio in metabolic stress conditions including hypoxia and glucose deprivation. Lately, many novel pathways, besides AMPK induction, have been revealed, which can explain some of metformin’s beneficial effects. It may help to identify new targets for treatment of diabetes and metabolic syndrome. Moreover, metformin is now attracting the attention of researchers in fields other than diabetes, as it has been shown to have anti-cancer, immunoregulatory and anti-aging effects. The aim of this review is to describe the potential anti-cancer and anti-aging properties of metformin and discuss the possible underlying mechanisms.

Both ZAP-70 and CD38 expression were shown to predict the clinical course of the disease, while ZAP-70 expression appeared to be more predictive than CD38 expression and more relevant in defining the cases of B-CLL responsive or refractory to first line chemotherapy. A simultaneous evaluation of ZAP-70 and CD38 expression allowed distinguishing the patients groups with the most favourable prognosis as well as those with the worst. Taken together we recommend assessing both ZAP-70 and CD38 protein expression for the definition of prognostic subgroups in patients with B-CLL.

Despite the presence of many hematological prognostic indexes, clinical course and overall survival are often highly variable even within the same patient subgroup. Recent studies suggest that simple, cost-effective, low-risk tests such as neutrophil to lymphocyte ratio (NLR) and lymphocyte to monocyte ratio (LMR) may be used to evaluate the prognosis. Their role has been well confirmed in diffuse large B-cell lymphoma (DLBCL), Hodgkin lymphoma (HL) and multiple myeloma (MM), but until now the prognostic significance of NLR and LMR in leukemias has not been widely reported. In this article, we analyze the literature data on prognostic value of NLR and LMR in haematological malignancies in the context of classic prognostic factors and clinical course.

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Cell synchronization is often achieved by inhibition of DNA replication. The cells cultured in the presence of such inhibitors as hydroxyurea, aphidicolin or thymidine become arrested at the entrance to S-phase and upon release from the block they synchronously progress through S, G 2 and M. We recently reported that exposure of cells to these inhibitors at concentrations commonly used to synchronize cell populations led to phosphorylation of histone H2AX on Ser139 (induction of γH2AX) through activation of ataxia telangiectasia mutated and Rad3-related protein kinase (ATR). These findings imply that the induction of DNA replication stress by these inhibitors activates the DNA damage response cell signaling pathways and caution about interpreting data obtained with the use of cells synchronized such way as representing unperturbed cells. The protocol presented in this chapter describes the methodology of assessment of phosphorylation of histone H2AX-Ser139, ATM/ATR substrate on Ser/Thr at SQ/TQ cluster domains as well as ataxia telangiectasia mutated (ATM) protein kinase in cells treated with inhibitors of DNA replication. Phosphorylation of these proteins is detected in individual cell immunocytochemically with phospho-specific Ab and measured by flow cytometry. Concurrent measurement of cellular DNA content and phosphorylated proteins followed by multiparameter cytometric analysis allows one to correlate extent of their phosphorylation with cell cycle phase.

B cell receptor (BCR) stimulation signal plays an important role in the pathogenesis of chronic lymphocytic leukemia (CLL), and kinase inhibitors directed toward the BCR pathway are now the promising anti-leukemic drugs. Ibrutinib, a Bruton tyrosine kinase inhibitor, demonstrates promising clinical activity in CLL. It is reported that ibrutinib, additionally to directly targeting leukemic cells, also inhibits the interactions of these cells with T cells, macrophages and accessory cells. Assessment of these mechanisms is important because of their non -direct anti-leukemic effects and to identify possible side effects connected with long-term drug administration.The aim of this study was to assess the in vivo effects of ibrutinib on T-cell subpopulations and cytokine network in CLL. The analysis was performed on a group of 19 patients during first month of ibrutinib therapy. The standard multicolor flow cytometry and cytometric bead array methods were used for assessment of T-cell subsets and cytokines/chemokines, respectively.The data obtained indicates that Ibrutinib treatment results in changes in T-cell subpopulations and cytokine network in CLL patients. Particularly, a significant reduction of T regulatory cells in peripheral blood was observed. By targeting these populations of T cells Ibrutinib can stimulate rejection of tumor cells by the immune system.

Red blood cell distribution width (RDW) is a quantitative measure of the variability in size of circulating erythrocytes. It was recently reported that RDW is a prognostic factor for infection diseases, cardiovascular and pulmonary diseases, as well as some neoplasms. Moreover, RDW is remarkably strong predictor of longevity, including all causes of death, for adults aged 45 years and older. To explain this occurrence it was proposed that persistent IGFs/mTOR signaling is one of the factors that play a role in affecting the RDW and mortality.The above observations induced us to analyze the prognostic role of RDW in chronic lymphocytic leukemia (CLL) being the most frequent type of adult leukemia in Western countries. The obtained results have shown that RDW may be considered as a potential CLL prognostic marker. Elevated RDW level at the moment of diagnosis was associated with advanced disease and presence of other poor prognostic factors. It is also connected with overall survival indicating shorter time in patients with elevated RDW. It is possible that the presently observed correlation between mortality and RDW of the CLL patients is affected by their metabolic (IGF-1/mTOR driven)- rather than chronological- aging. The patients with high level of RDW are expected to have an increased persistent level of IGF-1/mTOR signaling. Within the framework of personalized therapy, these CLL patients therefore would be expected to be more sensitive to the treatment with mTOR inhibitors.