Delfa Radić-Krišto

Notch pathway connections in primary leukaemia samples of limited size

BackgroundThe Notch pathway combined with other signalling molecules acts specifically for the development of each blood cell type and differentiation stage. A causative role of Notch dysfunction in leukaemia development has been found in many studies so, initially only for T- acute lymphoblastic leukaemia (T-ALL) but more recently also for B cell and myeloid leukaemia. The aim of our study is to introduce a method for multiple direct analysis of the Notch pathway partners in a population of only 500 or fewer cells. The notion of this method consists in gaining insight into gene expression at the level of the malignant clone population. A small number of cells is a significant limitation when working on primary cells either when freshly isolated or when analysed after several days in cocultures.MethodsThe primers were designed to avoid genomic amplification through the selection of 3′ and 5′ primers that hybridise with different exons. Cell lines and primary cells were collected and multiplex quantitative real-time reverse transcription polymerase chain reaction (qRT-PCR) performed on a descending number of cells, ranging from 2,500 cells up to 50 cells per sample, for the Notch pathway genes and other transcription factors important for cell differentiation. ImageJ program, STATISTICA 13.1 software package and Student’s t-test were used for statistical evaluation. We checked protein expression by western blot.ResultsWe characterised the gene expression levels of Notch, Ikaros and Parp genes in leukaemia cell lines of B and T origin and in primary leukaemia samples of limited size. We further compared our results to the cDNA analysis obtained by total RNA isolation from a large number of cells as routinely performed in clinical laboratories, and finally tested the method described on primary cells from leukaemia patients.ConclusionsThis rapid multiple gene expression analysis of a small population of cells provides efficient cell classification determining malignant changes as an important additional information for clinical leukaemia diagnostics as well as for in vitro studies of primary cells.

Rituximab Maintenance Strategy in Advanced Follicular Lymphoma: Facts and Controversies

Rituximab is a chimeric monoclonal CD20 antibody used in the treatment of CD20 positive non-Hodgkin lymphomas and has revolutionized treatment approach to these hematologic malignancies in the last decade. The main aim of this review is to present data on the use of rituximab in the treatment of follicular lymphoma (FL). We will focus on rituximab maintenance strategies in the first and second line treatment. This approach has improved the outcome in FL patients with better progression-free survival in all patients and better overall survival in relapsed setting. Regardless of good results, this strategy has generated controversies in medical community in the range from the lack of overall survival benefit in first line setting, adverse effects of possible overtreatment and toxicities to its unknown role in the era of novel agents. The existing data suggest that rituximab maintenance should be a rational therapeutic option for all patients with FL responding to fi rst line therapy and transplant-ineligible patients responding to reinduction.

Notch affects the prodifferentiating effect of retinoic acid and PMA on leukemic cells.

Notch proteins determine cell fate decisions in the development of diverse tissues. Notch has been initially found in T‐ALL but its role has been also studied in myelopoiesis and myeloid leukemias. Studies in different model systems have led to a widespread controversy as to whether Notch promotes or blocks myeloid differentiation. In this work, we evaluated the influence of Notch activation on leukemic cell differentiation along the monocytic and myelocytic pathway induced by phorbol 12‐myristate 13‐acetate (PMA) or all‐trans retinoic acid (ATRA). We observed that differentiation of the human myeloblastic cell line HL‐60 can be retarded or blocked by Delta/Notch interaction. ATRA induces complete remission in patients with acute promyelocytic leukemia, but it cannot completely eliminate the leukemic clone and to be effective it should be combined with chemotherapy. Our findings suggest that Notch signaling may contribute to the incomplete elimination of the leukemic cells after PMA or ATRA treatment and the blockage of Notch pathway may be beneficial in the treatment of myeloid leukemia.