Mati Shaklai

Serum CA 125 as a Prognostic factor in non-Hodgkin's lymphoma

Cancer antigen 125 (CA 125) is a glycoprotein expressed in normal tissues originally derived from coelomic epithelia such as peritoneum, pleura, pericardium, fallopian tubes and endometrium. Serum CA 125 levels are elevated in various benign and malignant conditions that involve stimulation of these tissues. Although elevated levels have been reported in patients with non-Hodgkins lymphoma (NHL), its role as a prognostic factor remained uncertain. In this study, serum CA 125 levels were measured prospectively in 108 consecutive patients with NHL: at diagnosis in 106, in remission in 39 and at relapse in 7. Levels were elevated in 43% at diagnosis. This finding was associated with advanced disease stage, bulky tumors, bone marrow involvement, extranodal disease (in stages III and IV), occurrence of B symptoms, pleural or peritoneal effusions, high serum LDH levels, high serum β2 microglobulin (β2-M) levels, elevated International Prognostic Score, poor performance status and partial or no response to treatment. No difference in CA 125 level was found between the indolent and aggressive lymphomas. Serum CA 125 levels at diagnosis had strong association with event-free and overall survival (<formula><italic>p</italic>=0.01</formula> and 0.003, respectively), with the patients with increased levels having worse survival. Patients with high CA 125 levels at diagnosis who achieved remission showed a significant decrease in CA 125 levels in remission. In conclusion, CA 125 is not only a reliable marker for staging and assessing tumor activity in NHL, elevated levels are also predictive of decreased survival.

Butyric acid and pivaloyloxymethyl butyrate, AN-9, a novel butyric acid derivative, induce apoptosis in HL-60 cells

A novel butyric acid derivative, pivaloyloxymethyl butyrate, AN-9, was previously shown to be a potent differentiating agent. AN-9 exerts a significant anticancer activity in vitro and in vivo. In all the activities examined, AN-9 was more potent than butyric acid. Here we show that AN-9 and butyric acid induce cell death by apoptosis. Exposure of HL-60 cells to butyric acid and AN-9 decreased cell numbers and induced cell differentiation and the appearance of typical apoptotic features. Induction of apoptosis and/or differentiation by AN-9 and butyric acid was dependent on the concentration and the time of exposure to the drugs. The advantage of AN-9 over butyric acid was further confirmed. Apoptosis induced by AN-9 occurred after a shorter exposure and at lower drug concentrations than that induced by butyric acid. Apoptosis by AN-9 was accompanied by reduction in Bcl-2 expression. Preincubation with antioxidants did not protect HL-60 cells from apoptosis induced by AN-9. HL-60 cells that were induced to differentiate by preincubation with retinoic acid or low AN-9 concentrations were more resistant to apoptosis, induced later by high concentrations of AN-9, than were undifferentiated cells.

Microvessel density in chemosensitive and chemoresistant diffuse large B-cell lymphomas

Preliminary reports involving a number of different kinds of tumors have indicated that microvessel quantification may be useful in predicting disease outcome. The aim of this study was to examine the relationship between microvessel density (MVD) as a parameter of tumor angiogenesis and the response to chemotherapy in diffuse large B-cell (DLBC) lymphomas.A total of 36 DLBC lymphoma patients were evaluated, 23 of them with a chemosensitive; responsive disease (median survival 8 y) and 13 with a chemoresistant, refractory disease (median survival 8 months). Microvessel quantification was performed by immunohistochemical staining, using monoclonal antibodies against factor VIII related antigen (F8RA) and against platelet/endothelial cell adhesion molecule-CD31.We found that F8RA stained a significantly higher number of blood vessels (about 2.5 times more) than CD-31; 7 samples were not stained with CD-31 but were positive for F8RA. There was no significant difference between the density of microvessel staining of the two groups. In the chemosensitive DLBC lymphomas positive for F8RA, the mean number of microvessels stained was 54.5±36.1 per microscopic field (200×) examined (range 6–149) whereas in the chemoresistant group the corresponding mean number was 43.1±25.5 (range 11–94).F8RA appears to be more sensitive for staining DLBC lymphomas microvessels than CD-31. Our data demonstrate that there is no correlation between tumor MVD and response to chemotherapy in patients with DLBC lymphomas.

Effect of the cytostatic butyric acid pro-drug, pivaloyloxymethyl butyrate, on the tumorigenicity of cancer cells

Previously we have shown that pivaloyloxymethyl butyrate (AN-9), a pro-drug of butyric acid (BA), is a differentiation-inducing agent in a variety of cells. In this report, we demonstrate that AN-9 is a cytostatic but not cytotoxic agent in a myelomonocytic cell line (WEHI); thus, the cells were growth-arrested and differentiated. These late changes in the cells were preceded by changes in the expression of the early regulatory genes,c-myc andc-jun. Although initiation of all these events had already occurred after 1 h exposure to AN-9, the tumorigenicity of these cells tested in Balb/c mice was not affected. A marked reduction in the tumorigenicity of AN-9-treated cells was observed after 4 h of exposure. Exposure of the highly metastatic subclone of Lewis lung carcinoma (3LLD122) to AN-9 resulted in a very pronounced effect on the tumorigenicity of these cells tested in C57BL mice. Unlike WEHI cells, the tumorigenicity of 3LLD122 was almost completely diminished after 1 h of exposure. In both cell types a 10-fold higher concentration of BA did not affect the tumorigenicity of the cells as did AN-9.

Significance of BCR-ABL Transcripts in Bone Marrow Aspirates of Philadelphia-Negative Essential Thrombocythemia Patients

Philadelphia-negative (Ph-neg) essential thrombocythemia (ET), polycythemia vera (PV) and idiopathic myelofibrosis (IMF) form a syndrome of related chronic myeloproliferative disorders (MPD) characterized by expansion of one or more of the hematopoietic progenitors. Based on our previous finding of BCR-ABL transcripts in bone marrow aspirates of 12/25 Ph-neg ET patients, we have expanded our study up to 40 patients. Here we describe the rational for performing this study and report 19 of 40 patients who have BCR-ABL transcripts in their BM, 11 of them carry b3a2 and 8 carry b2a2. The two groups, BCR-ABL positive and negative, were completely identical with regard to clinical characteristics and laboratory data. We also report preliminary results of our attempt to examine concordance or discordance of BCR-ABL expression in the peripheral blood and bone marrow of Ph-neg ET patients.

Doxorubicin and a butyric acid derivative effectively reduce levels of BCL-2 protein in the cells of chronic lymphocytic leukemia patient

Abstract: B‐chronic lymphocytic leukemia (B‐CLL) is a disease caused primarily by defects in the apoptosis mechanism. AN‐9, a butyric acid (BA) derivative, is a potent differentiating and an anti‐cancer drug that induces apoptosis in HL‐60 cells. Herein we show the affect of AN‐9, alone and in combination with doxorubicin, on cell cultures from B‐CLL patients. Cells from 17 patients were cultured and tested for viability, apoptosis, bcl‐2 and bax protein expression. Exposure of B‐CLL cell cultures to AN‐9 was accompanied by apoptosis and a marked viability loss (up to 46%, p=0.0017). AN‐9 reduced up to 51% (p=0.0017) the levels of bcl‐2 in 57% of the cultures that express bcl‐2. The combination of low concentrations of AN‐9 and doxorubicin more than additively enhanced apoptosis and reduced bcl‐2 levels in B‐CLL cultures which were resistant to AN‐9. AN‐9 enhanced bax expression up to 58%(p=0.008) in cultures from 53% of the patients, but had no effect on bax levels when combined with doxorubicin.

Detection of methylated ABL1 promoter in philadelphia-negative myeloproliferative disorders

Aberrant methylation of tumor-suppressor gene promoter regions may play a causal role in the pre-neoplastic stage of cancer progression. In chronic myeloid leukemia, changes in the methylation status of the CpG-rich islands at several sites in the proximal ABL1 promoter (Pa) on the Philadelphia (Ph)-chromosome have been observed. It remains unclear if the Pa methylation precedes the translocation event (t9;22) that generates the Ph-chromosome or if Pa methylation is a stochastic event in a progenitor cell which will later acquire other mutations, namely t9;22. The present study was conducted to answer two questions: What is the methylation status of Pa in patients with Ph-negative myeloproliferative disorders (MPD)? Can the study of methylation in patients with Ph-negative MPD shed light on the initial events associated with the translocation? To probe CpG methylation, we used two methodologies; site-methylation-sensitive restriction enzyme assay and methylation-specific PCR analysis following modification of genomic DNA by bisulfite. Results showed that 22 of the 97 patients with Ph-negative MPD expressed BCR-ABL transcripts. Seven of the 97 patients possessed methylated Pa, but only 2 of them expressed BCR-ABL transcripts. In some of the patients, Pa methylation was a dynamic event. In conclusion, aberrant methylation in Ph-negative MPD could be an initial event triggering the occurrence of the t9;22 translocation and its clinical expression. These findings may shed light on the pathogenesis and progression of MPD.

Levels of proteins C and S do not decline subsequent to first line chemotherapy in lymphoma patients

Thromboembolic complications and decrease in protein C and S have been observed in patients while receiving combination chemotherapy for breast cancer. We investigated whether initial cytotoxic treatment of non‐Hodgkins lymphoma (NHL) and Hodgkins disease (HD) is also associated with changes in these anticoagulant parameters. For this purpose 25 patients with intermediate to high grade NHL and seven with HD, undergoing primary treatment with cytotoxic drugs were evaluated at three time‐points: pre‐therapy, mid‐therapy and post‐therapy. In contrast to the breast cancer patients, no significant changes in protein C, protein S and antithrombin III levels were observed in the NHL patients during the various stages of therapy. However in HD patients, the mean protein C values had a tendency to be higher at mid‐therapy compared to pre‐therapy and protein S levels had a tendency to be higher at mid‐therapy compared to post‐therapy. In lymphoma patients receiving primary cytotoxic treatment we did not find changes in anticoagulant parameters that can explain a chemotherapy‐induced hypercoagulable state, as has been reported in breast cancer patients.