Cancer immunotherapy in mice and humans: personal observations

An experiment involving active immunotherapy of leukemia in mice and its extension to human patients withchronic lymphocytic leukemia is described. First, anticancer activation of the immune system, how it is achieved,and observations of the central blockade of the immune system in leukemia are discussed. Clinical results ofantileukemia immune activation are then presented, along with a description of new attempts to remove theimmune checkpoint blockade. Personal observations are presented on possible further steps to achieving moreeffective active immunotherapy of cancer

Modification of immunocytochemical ZAP-70 assay for potential clinical application in B-cell chronic lymphocytic leukemia

The ZAP-70 protein is a member of the Syk/ZAP protein tyrosine kinase family, normally expressed in T cells and NK cells but not found in normal, mature B cells. The protein plays a critical role in the initiation of T-cell signaling. Leukemic cells from patients with B-cell chronic lymphocytic leukemia (B-CLL) that expressed nonmutated immunoglobulin V genes were found to express levels of ZAP-70 protein that were comparable to those detected in T cells of healthy adults. The ZAP-70 protein expression can be evaluated by flow cytometry and may be used as a prognostic marker in B-CLL patients. We modified the method of immunocytochemical assessment of ZAP-70 expression. The traditional two-step method with monoclonal anti-ZAP-70 antibody in the first step followed by FITC-conjugated goat anti-mouse IgG was changed for one-step method with monoclonal anti-ZAP-70 antibody labeled by Zenon Alexa Fluor 488. The method is simple and fast. The major advantage of Zenon labeling technique is its compatibility with simultaneous staining of surface antigens. The cells may be earlier immunostained for CD3, CD19 and/or CD5 to compare of the ZAP-70 kinase expression in B and T cells

NK cell depletion and recovery in SCID mice treated with anti-NK1.1 antibody.

The anti-NK1.1 antibody produced by PK136 hybridoma cell line administered subcutaneously to SCID mice effectively decreased the level of peripheral blood NK cells and weight of the spleen for 3-4 days. The antibody treatment did not harm the general state of the animal, and may be practically applied in xenograft experiments

Characterization of a new small cell lung cancer (SCLC) cell line STP54 derived from a metastatic bioptate of a combined type of SCLC with Non-SCLC component.

Small cell lung cancer constitutes 15-20% cases of lung cancers, currently the leading cause of death from malignant diseases. It also causes the demise of >90% of affected individuals in 5 years. We have established a new SCLC cell line STP54 derived from fine needle aspirate of metastatic supraclavicular lymph node of 54 -year-old women for model experiments. The primary tumor was diagnosed by histopathological examination as combined type of small cell lung cancer with a non-small cell component. We cultured the cancer cells in the RPMI 1640 medium. In the long-term culture only the small cell component survived. The cell line was established after 30 passages and then characterized by performing cell morphology, cell growth analysis, tumorigenicity in vitro and flow cytometry analysis of selected markers (like NCAM, cytokeratines, HLA-ABC, Fas, Bcl-2, p53, CXCR4, CD210). The cells were growing in floating aggregates and show features suggesting its invasiveness. We suggest that this new cell line may serve as a valuable tool for further studies on lung tumor biology, molecular pathogenesis and metastatic mechanism

Monitoring cell proliferation in vitro with different cellular fluorescent dyes

 There are few methods for quantifying cell proliferation. Those tests describe the proliferation kinetics of a cell population, but they do not report the history of single cells, the number and frequency of cell divisions, or the precursor cell frequency. Cell-tracking assays based on dilution of the green fluorescent protein labelling dye, CFSE, has become the standard for monitoring cell proliferation. Other labelling dyes, e.g. CellTrace Violet and CellVue Claret, are also used for the same purpose. This study aimed to compare these three cell labelling methods for analysing the kinetics of cell viability, proliferation, and precursor cell frequency. Human peripheral blood mononuclear cells stimulated with Concanavalin A (ConA) were used as a model system. After labelling with a cell-tracking dye cells were divided into groups with and without ConA stimulation. From the 5th to 8th day, cells were collected and analysed with flow cytometry. Cell viability was not significantly different between labelled and unlabelled cells that received ConA stimulation. The proliferative fraction, proliferation index, and nonproliferative fraction were not significantly different among lymphocytes labelled with different dyes. Precursor cell frequency was also similar among cells labelled with the three cell-tracing dyes. The practical conclusion from our observations is that the results from cells labelled with different tracers may be compared directly and discussed jointly.

Patogeneza przewlekłej białaczki szpikowej - od genu do terapii celowanej

The occurrence of chronic myeloid leukemia (CML) is related to the appearance of reciprocal chromosomal translocation (9;22)(q34;q11). As a result of translocation between chromosomes 9 and 22, shortened chromosome 22 - Philadelphia (Ph) chromosome arises, coding for constitutively active protein tyrosine kinase, BCR-ABL1. The subsequent deregulation of the ABL1 kinase activity in cells leads to enhanced proliferation, resistance to apoptosis and altered adhesion. The discovery of imatinib, a relatively specific tyrosine kinase inhibitor, including ABL1 and BCR-ABL1 kinases, was a breakthrough in CML treatment. Imatinib functions by binding to the active site of ABL1 kinase in an inactive conformation, thus blocking the ATP binding site crucial for enzyme activity. Although imatinib has revolutionized CML therapy, appearance of resistance to this inhibitor leads to CML progression. In case of imatinib-resistance, several possible strategies exist for the management of the disease, which include increasing the dose of imatinib, changing therapy to the 2nd generation tyrosine kinase inhibitors or allogeneic hematopoietic stem cell transplantation. CML therapy, appearance of resistance to this inhibitor leads to CML progression. In case of imatinib-resistance, several possible strategies exist for the management of the disease, which include increasing the dose of imatinib, changing therapy to the 2nd generation tyrosine kinase inhibitors or allogeneic hematopoietic stem cell transplantation.CML therapy, appearance of resistance to this inhibitor leads to CML progression. In case of imatinib-resistance, several possible strategies exist for the management of the disease, which include increasing the dose of imatinib, changing therapy to the 2nd generation tyrosine kinase inhibitors or allogeneic hematopoietic stem cell transplantation. Hematologia 2010; 1, 3: 195-218Rozwój przewlekłej białaczki szpikowej (CML) jest związany z pojawieniem się wzajemnej translokacji chromosomalnej (9;22)(q34;q11). W wyniku wymiany fragmentów chromosomów 9 i 22 powstaje skrócony chromosom 22 - chromosom Philadelphia (Ph), który niesie nowy gen, kodujący konstytutywnie aktywną fuzyjną kinazę BCR-ABL1. Związana z tym zmieniona aktywność kinazy ABL1 prowadzi do zwiększonej proliferacji, oporności na apoptozę i zaburzonej adhezji komórek. Przełom w leczeniu CML nastąpił wraz z odkryciem specyficznego inhibitora kinazy tyrozynowej, w tym ABL1 i BCR-ABL1 - imatynibu. Imatinib hamuje aktywność kinazową BCR-ABL1, przyłączając się do miejsca aktywnego kinazy ABL1 w konformacji nieaktywnej i tym samym uniemożliwiając przyłączenie cząsteczki ATP, co jest niezbędne, by enzym był aktywny. Jakkolwiek terapia CML z użyciem imatynibu jest wysoce efektywna, pojawienie się u wielu pacjentów oporności na ten inhibitor prowadzi do progresji choroby. W przypadku stwierdzenia oporności na imatynib pomoc w pokonaniu oporności i uzyskaniu poprawy terapeutycznej mogą przynieść zwiększenie dawki imatynibu, zmiana leczenia na inhibitory kinazy tyrozynowej II generacji lub allogeniczne przeszczepienie macierzystych komórek krwiotwórczych. Hematologia 2010; 1, 3: 195-21