Adina Aviram

Ovarian minimal residual disease in chronic myeloid leukaemia

The options for fertility preservation include cryopreservation of ovarian tissue. Although transplantation of cryopreserved-thawed ovarian tissue in cancer survivors has resulted in live births, there is evidence of malignancy involvement in ovarian tissue, especially in leukaemia. The objectives of this study were to investigate the involvement of chronic myeloid leukaemia (CML) in ovaries by both pathological/immunohistochemical methods and PCR for the identification of the Philadelphia chromosome (BCR-ABL transcripts). The patient was a survivor of paediatric CML whose ovaries were cryopreserved. The patient became infertile and requested ovarian reimplantation in adulthood. Pathological examinations of ovarian tissue with immunohistochemical stainings, quantitative PCR and two-step nested PCR were applied to identify BCR-ABL transcripts. Despite the lack of positive pathological/immunohistochemical evidence, PCR and two-step nested PCR revealed that the ovary was contaminated by malignant minimal residual CML. Survivors of childhood CML may harbour minimal residual disease in the ovaries. This finding stresses the danger of reseeding cancer by ovarian grafting, especially in patients with leukaemia. If ovarian grafting is considered, reimplantation should be preceded by examination of ovarian samples both pathologically and by molecular techniques. On the basis of molecular findings, ovarian autografting was not recommended in this case report.

Uptake of pivaloyloxymethyl butyrate into leukemic cells and its intracellular esterase-catalyzed hydrolysis

Abstract Pivaloyloxymethyl butyrate (AN-9), a butyric acid (BA) prodrug, exhibited low toxicity and significant anticancer activity in vitro and in vivo. The purpose of this study was to elucidate the basis for AN-9 increased anticancer activity compared to BA, by studying the uptake of BA and AN-9 into the cells. Methods: The uptake rate and level of [14C]-AN-9 and [14C]-BA, labeled on the carboxylic moiety of BA, into HL-60 and MEL leukemic cell lines was measured. The cells were filtered and the retained radioactivity was determined. The dependence of the uptake on the activity of cellular esterases and membrane fluidity was investigated. Results: The uptake level in cells incubated with [14C]-AN-9 increased rapidly, peaked after 30 min in MEL and 1 h in HL-60 cells, and declined thereafter. This decline could be attributed to the hydrolysis of AN-9 by cellular esterases and catabolism of the released BA to CO2. In cells pretreated with an esterase inhibitor and incubated with [14C]-AN-9, the reduction of radioactivity was less precipitous. In cells exposed to [14C]-BA, the intracellular radioactivity level was low and unaffected by treatment with an esterase inhibitor. The uptake of [14C]-AN-9 decreased significantly at 4 °C compared to that at 37 °C. Conclusion: The higher potency of AN-9 compared to BA could be at least partially attributed to the more rapid uptake of the lipophilic AN-9 and the release of BA in the cells.

Fluoride-mediated activation of guinea pig neutrophils.

In guinea pig peritoneal neutrophils NaF at a concentration of above 5 mM elicited a dose-dependent, delayed and sustained activation of NADPH oxidase. Unlike in human neutrophils, in guinea pig cells, this response was independent of extracellular calcium. Fura2 fluorescence measurements indicated also a fluoride-mediated moderate elevation in the level of cytosolic calcium concentration. Pretreatment of neutrophils with pertussis toxin, blocked fluoride-promoted activation of NADPH oxidase, indicating that NaF stimulation was mediated by a G protein which is a pertussis toxin substrate. NaF-elicited calcium elevation was insensitive to the toxin. Upon transfer of NaF-stimulated cells to a fluoride-free medium, superoxide release declined and calcium levels diminished. The response of the deactivated, fluoride-prestimulated guinea pig neutrophils to a secondary stimulation with phorbol myristate acetate (PMA) or fMet-Leu-Phe, was either unaffected by the previous challenge with NaF (PMA) or augmented by it (the chemotactic peptide). In parallel to the activation of NADPH oxidase, NaF also induced translocation of protein kinase C to cell membranes. This effect was also abolished by a pretreatment with pertussis toxin.

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.

Follicular Lymphoma with Extensive Gastrointestinal Tract Involvement: Follow-up by Capsule Endoscopy.

Follicular lymphoma with gastrointestinal tract involvement is rare. We describe the case of a young woman with follicular lymphoma with multiple nodular lesions involving segments of the proximal jejunum and terminal ileum. The presenting symptom was chronic diarrhea. The diagnosis was made by endoscopy with histologic examination of the mucosal lesions of the proximal and distal small intestine, immunohistochemical staining, and molecular analysis. The initial spread and pattern of the small bowel involvement, as well as treatment response, were evaluated by videocapsule endoscopy. The application of molecular analysis along with immunophenotypic evaluation has made it possible to precisely diagnose follicular lymphoma. In the present case, the use of capsule endoscopy improved the evaluation of the extent of small bowel involvement prior to and following treatment.

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.

Differentiation-associated changes of cation-transport activities in myeloid leukemic cell lines

Induction of differentiation in HL‐60 and U‐937 leukemic cell lines, resulted in 1.5–10‐fold increase in 45Ca2+ uptake. The increased 45Ca2+ uptake in the differentiating cells was inhibited by verapamil, cromolyn and amiloride. Elevation in Ca2+ uptake in differentiating cells was also demonstrated using the fluorescent probe, fura‐2 acetoxymethyl ester. The increased 45Ca2+ uptake was accompanied by a decrease in ouabain‐insensitive and ‐sensitive 86Rb+ uptake. Furthermore, correlation between the changes in these activities was observed. Modulation of extracellular pH affected differentiation: higher pH increased the extent of differentiation.

Acute promyelocytic leukemia with isochromosome 17q and cryptic PML-RARA successfully treated with all-trans retinoic acid and arsenic trioxide.

Acute promyelocytic leukemia (APL) is a subtype of acute leukemia that is characterized by typical morphology, bleeding events and distinct chromosomal aberrations, usually the t(15;17)(q22;q21) translocation. Approximately 9% of APL patients harbor other translocations involving chromosome 17, such as the t(11;17)(q23;q21), t(5;17)(q35;q12-21), t(11;17)(q13;q21), and der(17). All-trans retinoic acid (ATRA) and arsenic trioxide (ATO) have specific targeted activities against the PML-RARA fusion protein. The combination of ATRA and ATO is reportedly superior to chemotherapy and ATRA as induction therapy for APL. The clinical significance of non-t(15:17) APL-related aberrations is controversial, with conflicting reports regarding sensitivity to modern, targeted therapy. Isochromosome 17q (iso(17q)) is rarely associated with APL and usually occurs concurrently with the t(15:17) translocation. No published data is available regarding the efficacy of ATO-based therapy for APL patients who harbor iso(17q). We report on an APL patient with iso(17q) as the sole cytogenetic aberration and a cryptic PML-RARA transcript, who was treated with ATRA and ATO after failure of chemotherapy and achieved complete remission. To our knowledge, this is the first published report of APL associated with iso(17q) as the sole cytogenetic aberration, which was successfully treated with an ATO containing regimen.