Merav Leiba

Phase I Safety and Pharmacokinetic Study of CT-011, a Humanized Antibody Interacting with PD-1, in Patients with Advanced Hematologic Malignancies

Purpose: CT-011 is a humanized IgG1 monoclonal antibody that modulates the immune response through interaction with PD-1, a protein belonging to the B7 receptor family present on lymphocytes. The objectives of this phase I study were to assess the dose-limiting toxicities, to determine the maximum tolerated dose, and to study the pharmacokinetics of CT-011 administered once to patients with advanced hematologic malignancies. Experimental Design: Seventeen patients were treated with escalating doses of CT-011 ranging from 0.2 to 6 mg/kg. For pharmacokinetic analysis, blood samples were withdrawn from the patients before and immediately after treatment and at 24 hours, 48 hours, and on days 7, 14, and 21. CT-011 blood levels were assessed with a specific ELISA and derived concentrations were used to calculate pharmacokinetic parameters. Activation of the immune system was assessed by measuring peripheral blood CD4+, CD8+, and CD69+ lymphocytes. Results: The study showed the antibody to be safe and well tolerated in this patient population. No single maximum tolerated dose was defined in this study. Clinical benefit was observed in 33% of the patients with one complete remission. Pharmacokinetic analyses show that serum Cmax and the AUC of CT-011 increased proportionally with dose. The median t1/2 of CT-011 ranged from 217 to 410 hours. Sustained elevation in the percentage of peripheral blood CD4+ lymphocytes was observed up to 21 days following CT-011 treatment. Conclusions: A single administration of 0.2 to 6.0 mg/kg of CT-011 is safe and well tolerated in patients with advanced hematologic malignancies.

Lenalidomide targets clonogenic side population in multiple myeloma: pathophysiologic and clinical implications

Recurrence of multiple myeloma (MM) after therapy suggests the presence of tumor-initiating subpopulations. In our study, we performed flow cytometry-based Hoechst 33342 staining to evaluate the existence of a MM population with stem-like features known as side population (SP) cells. SP cells exhibit substantial heterogeneity in MM cell lines and primary MM cells; express CD138 antigen in MM cell lines; display higher mRNA expression and functional activity of ABCG2 transporter; and have a higher proliferation index compared with non-SP cells. We observed evidence for clonogenic potential of SP cells, as well as the ability of SP cells to regenerate original population. Moreover, SP cells revealed higher tumorigenicity compared with non-SP cells. Importantly, lenalidomide decreased the percentage and clonogenicity of SP cells, and also induced phosphorylation changes in Akt, GSK-3α/β, MEK1, c-Jun, p53, and p70S6K in SP cells. Adherence to bone marrow stromal cells (BMSCs) increased the percentage, viability, and proliferation potential of SP cells. Lenalidomide and thalidomide abrogated this stimulatory effect of BMSCs and significantly decreased the percentage of SP cells. Our studies demonstrate a novel mechanism of action for lenalidomide, namely targeting SP fraction, providing the framework for new therapeutic strategies targeting subpopulations of MM cells including presumptive stem cells.

Anti-tumor activity and signaling events triggered by the isothiocyanates, sulforaphane and phenethyl isothiocyanate, in multiple myeloma.

Background Isothiocyanates, a family of phytochemicals found in cruciferous vegetables, have cytotoxic effects against several types of tumor cells. Multiple myeloma is a fatal disease characterized by clonal proliferation of plasma cells in the bone marrow. The growing body of preclinical information on the anti-cancer activity of isothiocyanates led us to investigate their anti-myeloma properties. Design and Methods We evaluated the anti-myeloma activity of the isothiocyanates, sulforaphane and phenethyl isothiocyanate, on a panel of human myeloma cell lines as well as primary myeloma tumor cells. Cell viability, apoptosis, cell cycle alterations and cell proliferation were then analyzed in vitro and in a xenograft mouse model in vivo. The molecular sequelae of isothiocyanate treatment in multiple myeloma cells were evaluated by multiplex analyses using bead arrays and western blotting. Results We observed that sulforaphane and phenylethyl isothiocyanate have activity against myeloma cell lines and patients‘ myeloma cells both in vitro and in vivo using a myeloma xenograft mouse model. Isothiocyanates induced apoptotic death of myeloma cells; depletion of mitochondrial membrane potential; cleavage of PARP and caspases-3 and -9; as well as down-regulation of anti-apoptotic proteins including Mcl-1, X-IAP, c-IAP and survivin. Isothiocyanates induced G2/M cell cycle arrest accompanied by mitotic phosphorylation of histone H3. Multiplex analysis of phosphorylation of diverse components of signaling cascades revealed changes in MAPK activation; increased phosphorylation of c-jun and HSP27; as well as changes in the phosphorylation of Akt, and GSK3α/β and p53. Isothiocyanates suppressed proliferation of myeloma cells alone and when co-cultured with HS-5 stromal cells. Sulforaphane and phenylethyl isothiocyanate enhanced the in vitro anti-myeloma activity of several conventional and novel therapies used in multiple myeloma. Conclusions Our study shows that isothiocyanates have potent anti-myeloma activities and may enhance the activity of other anti-multiple myeloma agents. These results indicate that isothiocyanates may have therapeutic potential in multiple myeloma and provide the preclinical framework for future clinical studies of isothiocyanates in multiple myeloma.

Prior treatment with the tyrosine kinase inhibitors dasatinib and nilotinib allows stem cell transplantation (SCT) in a less advanced disease phase and does not increase SCT Toxicity in patients with chronic myelogenous leukemia and philadelphia positive acute lymphoblastic leukemia

Prior treatment with the tyrosine kinase inhibitors dasatinib and nilotinib allows stem cell transplantation (SCT) in a less advanced disease phase and does not increase SCT Toxicity in patients with chronic myelogenous leukemia and philadelphia positive acute lymphoblastic leukemia

Philadelphia-Chromosome-Positive T-Lymphoblastic Leukemia: Acute Leukemia or Chronic Myelogenous Leukemia Blastic Crisis

The Ph1 chromosome has rarely been reported in T-lineage acute lymphoblastic leukemia (T-ALL), and the clinical relevance of this translocation in T-ALL is currently unknown. In chronic myelogenous leukemia (CML) some data indicate derivation of T-cells from the leukemic clone and only a few cases of T-derived blastic crisis have been reported and quite often disputed. Particularly in cases identified initially in blastic crisis it may be difficult to distinguish those from Ph1-positive T-ALL. We herein report 2 patients who presented with a clinical picture of Ph1-positive T-ALL and who raised a differential diagnosis from T-cell blastic crisis of CML. We review the literature and suggest clinical and laboratory features that can help in the diagnosis. According to our literature review, 23 cases of Ph1-positive T-ALL and 44 cases of T-cell blastic crisis of CML, including ours, were reported. Some major differences between the two entities could help in establishing a diagnosis of Ph1-positive T-cell blastic crisis of CML vs. Ph1-positive T-ALL: Male sex and younger age was more predominant in T-ALL. While in most cases of CML blastic crisis there was a history of CML there was no such history in the T-ALL cases. Medullary involvement with lymphoblastic leukemia was present in all cases of T-ALL but only in about half of the cases of CML blastic crisis. None of the CML-blastic crisis cases tested by RT-PCR showed the minor breakpoint transcript, while 2 cases with T-ALL had the minor breakpoint transcript and 1 had both transcripts. Combined morphologic and FISH analysis can help to distinguish between the two entities and was applied in one of our cases. Although both entities carry a severe prognosis, differentiating between them might have clinical relevance, especially in the imatinib era.

Association of heparanase gene (HPSE) single nucleotide polymorphisms with hematological malignancies

Heparanase, endo-β-D-glucuronidase, degrades heparan sulfate glycosaminoglycans – the principal polysaccharide of the basement membrane and extracellular matrix. Heparanase activity plays a decisive role in biological processes associated with remodeling of the extracellular matrix, such as cancer metastasis, angiogenesis and inflammation. In the hematopoietic system, heparanase is thought to be associated with normal differentiation and function of myeloid cells and platelets. We investigated heparanase polymorphisms in patients with acute myeloid leukemia (AML), myelodysplastic syndrome (MDS), acute lymphoblastic leukemia (ALL), chronic myeloid leukemia (CML), Hodgkins disease (HD) and multiple myeloma (MM). Significant correlation was found between rs11099592 and rs6535455 heparanase gene (HPSE) single nucleotide polymorphisms (SNPs) and ALL (χ21d.f.=4.96, P=0.026). Genotype frequency comparisons revealed a significant association with rs4693602 (χ22d.f.=7.276, P=0.026) in MM patients and rs4364254 (χ22d.f.=6.226, P=0.044) in AML patients. Examination of HPSE gene mRNA expression by real-time RT-PCR indicated a significant low HPSE gene expression level in ALL patients and a high expression level in MM and AML patients, compared to healthy controls. Moreover, statistically significant correlation was found between heparanase mRNA expression level and three HPSE gene SNPs (rs4693608, rs11099592 and rs4364254) among healthy individuals. These data suggest that certain HPSE gene SNPs may contribute to basal heparanase gene expression and that alterations in this gene are an important determinant in the pathogenesis of ALL, AML and MM.

Urinary hepcidin excretion in patients with myelodysplastic syndrome and myelofibrosis

Many patients with myelodysplastic syndrome (MDS) develop severe anaemia that requires multiple blood transfusions with consequent iron overload. The absorption of iron from the intestine, iron release from hepatic stores and the recycling of iron by macrophages is negatively regulated by hepcidin, the principal iron-regulatory hormone (Nicolas et al, 2002; Ganz & Nemeth, 2006). Hepcidin regulates iron absorption and distribution by binding to ferroportin, the iron exporter of these cells, and inducing its internalization and degradation, (Nemeth et al, 2004). Hepcidin is increased in response to increased body iron levels, transferrin saturation (Ganz & Nemeth, 2006) or inflammation, and is decreased in response to hypoxia, (Nicolas et al, 2002) erythropoietic activity (Adamsky et al, 2004) and oxidative stress (Choi et al, 2007). Patients with congenital haemolytic anaemia, such as thalassaemia, develop severe iron overload both due to multiple blood transfusions and to increased iron absorption. Hepcidin levels are inappropriately decreased in thalassaemia intermedia, whereas in thalassaemia major, hepcidin levels were found to be elevated, presumably due to transfusions which decrease erythropoietic drive (Origa et al, 2007). The present study investigated whether urinary hepcidin levels are also low in patients with various forms of MDS, similar to the findings in thalassaemia intermedia. Approval was obtained from the University of California, Los Angeles (UCLA) and Wolfson Medical Centre institutional review boards for the study. Urine samples were collected during visits to the outpatient department of the Wolfson Medical Centre. The patients lacked any clinical signs of inflammation. Urine samples were obtained from four patients with refractory anaemia (RA), four with refractory anaemia with ringed sideroblasts (RARS), three with refractory cytopenia with multilineage dysplasia (RCMD), two with refractory anemia with excess of blasts I (RAEB I), four with refractory anaemia with excess of blasts II (RAEB II) and four with idiopathic myelofibrosis (MF), with an age range from 54 to 87 years (Table I). Nine patients had received less than 10

Cytogenetics of extramedullary manifestations in multiple myeloma

Extramedullary disease in patients with multiple myeloma is a rare event, occurring mostly in advanced disease or relapse. Outcome is poor and prognostic factors predicting the development of extramedullary disease have not been defined. We investigated cytogenetic alterations of myeloma cells in different extramedullary manifestations by adapting the fluorescence in situ hybridization (FISH) technique in combination with cytoplasmic immunoglobulin staining to study the cytogenetics of plasma cell tumours on paraffin embedded material. Thirty six patients were investigated: 19 with extramedullary disease, 11 with skeletal extramedullary disease and six with solitary extramedullary plasmacytoma. The first two groups showed the following results: del(17p13) 32% vs. 27%, del(13q14) 35% vs. 27%, MYC‐overrepresentation 28% vs. 18% and t(4;14) 37% vs. 18%. We detected an overall higher incidence of del(17p13) in both groups compared to data from bone marrow samples of multiple myeloma reported to date (range 7–16%). The solitary extramedullary plasmacytomas presented overall less cytogenetic aberrations than the other groups. Most important, three patients with extramedullary disease and one with skeletal extramedullary disease presented different FISH findings in the extramedullary tumour compared to their bone marrow plasma cells. del(17p13), occurring additional in three of four cases, seems a strong marker for extramedullary progression of myeloma.

Changes in Gene Expression Pattern following Granulocyte Colony-Stimulating Factor Administration to Normal Stem Cell Sibling Donors

Granulocyte colony-stimulating factor (G-CSF) is widely used for the mobilization of hematopoietic stem cells from normal donors. The mechanism of induced mobilization has recently been elucidated. Treatment with G-CSF is considered safe; however, long-term effects are largely unknown. The aim of this study was, therefore, to determine whether it leads to significant changes in gene expression modifications. Affymetrix Gene Chip array technology was used. Samples were collected before and at various time points (up to 9 months) after mobilization. The expression levels of 122 genes were transiently modified before and after mobilization (p < 0.05). Fifty-three genes belonging to cell growth, proliferation and communication gene ontology categories were upregulated, while 69 genes were downregulated. Conclusion: The administration of G-CSF is associated with transient DNA and gene expression modifications in the lymphocytes of normal mobilized donors. A long-term follow-up of stem cell donors is recommended.

Characterization of HPSE Gene Single Nucleotide Polymorphisms in Jewish Populations of Israel

Heparanase is a mammalian endoglucuronidase responsible for heparan sulfate (HS) degradation. HS is a major constituent of the extracellular matrix (ECM) and HS-degrading activity plays a decisive role in fundamental biological processes associated with remodeling of the ECM, such as cancer metastasis, angiogenesis and inflammation. There is great interest in the prospect of genome-wide association studies to identify genetic factors underlying complex diseases. It is important to establish a detailed description of the heparanase (HPSE) gene single nucleotide polymorphisms (SNPs). In this study, four Israeli Jewish populations (Ashkenazi, North African, Mediterranean and Near Eastern) were examined for 7 HPSE gene SNPs. Four out of 7 SNPs (rs4693608, db11099592, rs4364254, db6856901) were found to be polymorphic. Population comparisons revealed significant differences in SNPs allele frequency between Near Eastern and each of the other three populations. Genotype and allele frequencies in Jewish populations were different from non-Jewish populations, except for a certain similarity to Caucasians. Although the distance between SNPs is relatively small, the db11099592 SNP was in linkage disequilibrium (LD) only with the proximal SNP rs4693608. LD between distal SNPs rs4364254 and db6856901 was found only in Mediterraneans and North Africans. The current study provides a characterization of the normally occurring HPSE gene SNPs in different populations. This information is obligatory for further studies on the linkage between these SNPs and heparanase expression and function in various pathological processes, primarily cancer progression.