ALBERT

Description: Introduction: Arsenic trioxide (AT) has impressive single agent activity in relapsed acute promyelocytic leukemia. It also has activity in myelodysplastic syndromes and multiple myeloma. In vitro data has suggested increased cytotoxicity when combined with agents that deplete intracellular glutathione, forming the rationale for combination studies. Ascorbic acid (AA) can deplete intracellular glutathione and may potentiate the cytotoxicity of AT. Upon this basis, we initiated a phase II study of arsenic trioxide plus ascorbic acid for relapsed/refracory lymphoid malignancies. Arsenic trioxide was administered at a dose of 0.25 mg/kg IV over one hour M-F for one week and then 2X/week for 5 weeks. Each arsenic infusion was followed by an infusion of 1000 mg ascorbic acid over 15 minutes. Each 6-week cycle was followed by a two-week rest period before repeating the cycle. Treatment was continued until best response plus two cycles or progressive disease. Patient characteristics: Median age 70.5 (37–88). Gender 10M, 6F. Histologies CLL/SLL (4), Follicular (3), Mantle Cell (3), DLBCL (2), Burkitt (2), Marginal Zone (1), Hairy Cell (1). Median # Prior therapies 4 (2–13). Refractory to prior treatment 13/16. Median ECOG PS 1 (0–2). B symptoms 3/16. Elevated LDH 8/16. Elevated B2M 13/16. Results: Median number of completed cycles 1 (0–4). Eight patients did not complete cycle #1, six due to progressive disease (PD) and 2 due to toxicity. Of the 2 patients coming off for toxicity, one patient with known coronary artery disease suffered a myocardial infarction on the 4th day of treatment and expired from congestive heart failure and the other experienced repeated grade 4 hyperglycemia. Six patients completed one cycle of therapy and were removed for PD. One patient completed 3 cycles of therapy before experiencing PD. One patient with mantle cell lymphoma received 4 cycles of therapy and achieved a CRu. The overall response rate was 6% (1/16). The responding patient’s treatment was stopped after 4 cycles for MD/patient preference and she experienced PD 5 months after completion of therapy. Grade 3 toxicities included thrombocytopenia (2 patients), anemia (3), neutropenia (1), stomatitis (1), anorexia (1), and elevated LFTs (1). Grade 4 toxicities included neutropenia (2) and hyperglycemia (1). Conclusions: AT plus AA in this dosing schedule had modest toxicity but limited antitumor activity. The data should be interpreted in the context of our heavily treated, essentially refractory patient population. Our trial had a two-stage design, and was closed due to lack of activity at the first stage analysis. Other doses and schedules may prove to be more efficacious.

Description: The nucleosome is the basic structure of chromatin. Changes in the biochemical composition of nucleosome-associated histone tails are associated with specific gene activation states, and are the target of several antineoplastic agents such as histone deacetylase inhibitors (HDI). Nucleosomes are constrained into loops that are flanked by domains known as matrix-attached regions (MARs). MARs contain DNA topoisomerase II (Topo II) consensus sequences. Topo II is responsible for regulating and maintaining DNA topology and is the target of several antineoplastic agents such as the anthracycline IDA, an effect mediated by the induction of double strand DNA breaks (DSB). We hypothesized that the combination of a Topo II inhibitor and a HDI will have synergistic antileukemia activity. VPA and SAHA are two HDIs currently studied in several clinical trials with known antileukemia activity and tolerable toxicity. To test our hypothesis and to develop future clinical studies, we have analyzed the effect of the combination of IDA, a potent Topo II inhibitor, with VPA or SAHA. We treated the leukemic cells lines MOLT4 and HL60 with increasing doses of IDA (0.5-20nM), SAHA (0.3-3μM) or VPA (0.25-3mM) daily for 3 days. First, using trypan blue viability assays, we identified the IC10 of IDA to be 0.5nM for MOLT4 and 1.5nM for HL60. Doses in excess of 2μM of SAHA or 3mM of VPA resulted in more than 90% decrease in cell viability in both cell lines. Subsequently, SAHA at doses of 0.075-1μM and VPA at 1-3mM were used for the combination experiments with IDA at its specific cell line IC10. At low doses of SAHA (0.075-0.45 μM) and VPA (0.25-1 mM) the combination was shown to have synergistic antileukemia activity by the Fractional Product Method of Webb. These results were confirmed using Annexin V assays. Of importance, growth inhibition was independent of the sequence used. To analyze the effects of this combination on DSB generation, we analyzed using immonocytochemistry and western blot, the induction of γH2AX, a histone variant that has been identified as an early event after the DSBs. SAHA alone induced a modest increase in γH2AX compared to baseline, whereas IDA alone had a significant effect that was not potentiated by the addition of SAHA. Histone H3 and H4 acetylation increased in a dose-dependent manner (2.4–15 fold) with both SAHA and VPA, starting at 0.3μM of SAHA and 0.25mM of VPA. The addition of IDA had no significant effect on histone acetylation. Because of previous data indicating that HDIs may down-regulate the expression of Topo II-alpha, the target of IDA, we have studied using real-time PCR its levels prior and during exposure to the different combinations. SAHA or VPA had no effect on Topo II-alpha mRNA levels whereas IDA induced 2.0–3.5 fold its expression in a dose-independent manner, an effect no altered by the addition of SAHA or VPA. Expression of p21CIP1, that is silenced in both cell lines, was restored by single agent VPA, SAHA or IDA. The combination of these drugs resulted in an additive effect in terms of p21CIP1 induction. Despite this phenomenon, no changes in cell cycle status were observed in these cells. In summary, the combination of IDA and SAHA or VPA has potent in vitro antileukemia effect, and should be studied in clinical trials.

Description: Introduction. The umbilical cord stem cell bank was created in Mexico City in june 2003 due to the need to have access to pluripotential stem cells to cover the hematological and immunological pediatric needs. We have so far 300 units of umbilical stem cells available to the medical community. The bank has been designed with a completely automatic process and the standards are based in NETCORD-FAHCT outlines. Objective. In the present study we made a balance of the evaluation of umbilical cord stem cell process including the maternal setting, the standarization of the methodology to obtain the stem cells, compared to other institutions around the world. Material and methods. Bayesian analysis allow us to evaluate our procedures at the different levels. Bayesian networks are directed acyclic graphs (DAGs) where the nodes are random variables and certain independence assumptions hold. Results. In table 1 we show the results of the first 300 units process with the automatic process. The arcs in a bayesian network specify the independence assumptions that must hold between the random variables and the global dependence of the total factors. Figure 1. Conclusion. Through the bayesian analysis, we found a direct influence of the collected volume, the time between the collection and the procedure, and the maternal unit, with respect to the number of recovered cells, specifically with CD34+ the viable ones as well as the totals. Figure 1. Bayesian Analysis between neonatal and process factors. Weight baby (PRN); sex (SRN); cord blood unit collected volume (ml); initial white cell (GBTI); total final white cell(GBTF); % total CD34+ ( PCD34T); % viable CD34+ (PCD34V); total CD43+ (CD34T); viable CD34+ (CD34VA); ginecology unit (UH); time hour (TH). Figure 1. Bayesian Analysis between neonatal and process factors. Weight baby (PRN); sex (SRN); cord blood unit collected volume (ml); initial white cell (GBTI); total final white cell(GBTF); % total CD34+ ( PCD34T); % viable CD34+ (PCD34V); total CD43+ (CD34T); viable CD34+ (CD34VA); ginecology unit (UH); time hour (TH). Results of the first 300 units process with these technology Initial nuclear cells/ml Collectionvolume Initial total nuclear cells % CD 34total Viable% CD 34 Recovery% Average 11.4 x 106/ml 102.8 ml 12.2 x108 0.32 0.31 80 Acceptance criterion 7–20 x 106 〉 75 ml 〉8 x 108 0.1– 0.3 0.1– 0.3 〉60%

Description: L-selectin is an adhesion molecule that plays an essential role in the early events of the inflammatory response. Our group has recently described that several nonsteroidal anti-inflammatory drugs (NSAIDs) are able to induce both in vivo and in vitro the shedding of L-selectin in neutrophils through an unknown mechanism. In this work, we have studied potential mechanisms involved in the shedding of L-selectin induced by NSAIDs. This effect of NSAIDs did not involve any detectable intracellular calcium flux. Pretreatment of neutrophils either with Ro 31-8220 and H7, 2 specific inhibitors of protein kinase C (PKC), or with inhibitors of protein tyrosine kinases such as tyrphostin A25 or herbimycin A did not prevent the NSAID-mediated L-selectin shedding. However, the KD-IX-73-4, an inhibitor of L-selectin proteolysis was able to block the effect of NSAIDs on L-selectin expression. Remarkably, NSAIDs caused a variable reduction in the neutrophil intracellular ATP concentration that highly correlated with the differential ability of NSAIDs to trigger L-selectin shedding (r = 0.8, P 

Description: Aberrant DNA methylation of promoter-associated CpG islands is a frequent phenomenon in human leukemias, and in particular in adult ALL. Hck is a member of the Src family of tyrosine kinases, and functionally is located downstream of BCR-ABL signaling in chronic myelogenous leukemia (CML). Hck expression is limitedly to myeloid cells and B cell lymphocytes. Although some evidence indicates that Hck is required for malignant transformation and apoptosis, its role in leukemia is not fully understood. Here we analyze the role of aberrant DNA methylation of Hck in leukemia cell lines and patients. Using BLAT, we first identified the presence of a canonical CpG island in the near proximity of the transcription start site of HcK. To detect and measure DNA methylation, we designed a combined bisulfite restriction PCR assay. Using this assay, we found that Hck was methylated in 13 out of 23 hematopoietic and 8 out of 10 non-hematopoietic cell lines, but not in the bone marrow from 6 healthy individuals. We subsequently studied Hck expression by real-time PCR using GAPDH expression as an internal control. Hck expression was lower (dCT = −14.2± 3.6) in 7 Hck methylated cell lines than in 8 Hck unmethylated ones (dCT= −9.0± 3.5), p=0.017. All the cell lines studied were of myeloid or B cell origin. We then treated the Raji cell line with the hypomethylating agent 5-aza-2-deoxycytidine (DAC). DAC treatment resulted in partial hypomethylation of Hck and in an increment of Hck expression (dCT: −19.37 to −8.47). Subsequently, the effects of DAC treatment on Hck protein expression levels were analyzed using Western blot. These experiments showed a strong correlation between hypomethylation, gene re-expression and protein expression levels. These data therefore indicates that DNA methylation is an important aberrant regulator of Hck expression in leukemia cell lines. Based on the relevance of these findings, we then analyzed the frequency of Hck methylation in patients with leukemia. Using a cut-off of 10%, Hck was found to be methylated in 15 out of 44 (34%) patients with ALL, 9 out 23 pts (39%) with CML, and 3 out 10 pts (30%) with AML. Of importance, the density of Hck methylation was significantly higher in patients with ALL (mean 11.3%; range 0–76) compared to those with CML(5.2%; range 0–12) or AML ( 7.5%, range 0–14), p=0.02. Hck methylation was not associated with a B cell phenotype or the presence of the Philadelphia chromosome in patients with ALL. Nine ALL pts out of 15 with Hck methylation had died compared to 7 out 29 unmethylated (total ALL group n=34). Median survival had not been reached for the group of patients with no Hck methylation (n=29) compared to 116 weeks for those with Hck methylation (n=15) (p=0.08). All pts had been treated with hyperCVAD based chemotherapy. These data indicates that Hck methylation is a frequent phenomenon in human leukemia that maybe associated with a worse prognosis in ALL and suggests that Hck has a tumor suppressor like function in these disorders.

Description: Endoglin is an endothelial membrane glycoprotein involved in cardiovascular morphogenesis and vascular remodeling. It associates with transforming growth factor-β (TGF-β) signaling receptors to bind TGF-β family members, forming a functional receptor complex. Arterial injury leads to up-regulation of endoglin, but the underlying regulatory events are unknown. The transcription factor KLF6, an immediate-early response gene induced in endothelial cells during vascular injury, transactivates TGF-β, TGF-β signaling receptors, and TGF-β–stimulated genes. KLF6 and, subsequently, endoglin were colocalized to vascular endothelium (ie, expressed in the same cell type) following carotid balloon injury in rats. After endothelial denudation, KLF6 was induced and translocated to the nucleus; this was followed 6 hours later by increased endoglin expression. Transient overexpression of KLF6, but not Egr-1, stimulated endogenous endoglin mRNA and transactivated the endoglinpromoter. This transactivation was dependent on a GC-rich tract required for basal activity of the endoglin promoter driven by the related GC box binding protein, Sp1. In cells lacking Sp1 and KLF6, transfected KLF6 and Sp1 cooperatively transactivated theendoglin promoter and those of collagen α1(I), urokinase-type plasminogen activator, TGF-β1, and TGF-β receptor type 1. Direct physical interaction between Sp1 and KLF6 was documented by coimmunoprecipitation, pull-down experiments, and the GAL4 one-hybrid system, mapping the KLF6 interaction to the C-terminal domain of Sp1. These data provide evidence that injury-induced KLF6 and preexisting Sp1 may cooperate in regulating the expression of endoglin and related members of the TGF-β signaling complex in vascular repair.

Description: We have identified a cell population expressing erythroid (TER-119) and megakaryocyte (4A5) markers in the bone marrow of normal mice. This population is present at high frequency in the marrows and in the spleens involved in the erythroid expansion that occurs in mice recovering from phenylhydrazine (PHZ)-induced hemolytic anemia. TER-119+/4A5+ cells were isolated from the spleen of PHZ-treated animals and were found to be blast-like benzidine-negative cells that generate erythroid and megakaryocytic cells within 24-48 hours of culture in the presence of erythropoietin (EPO) or thrombopoietin (TPO). TER-119+/4A5+ cells represent a late bipotent erythroid and megakaryocytic cell precursors that may exert an important role in the recovery from PHZ-induced anemia.

Description: This study aimed to correlate the frequency of somatic mutations in the IgVH gene and the use of specific segments in the VH repertoire with the clinical and characteristic features of a series of 35 cases of splenic marginal zone lymphoma (SMZL). The cases were studied by seminested polymerase chain reaction by using primers from the FR1 and JH region. The results showed unexpected molecular heterogeneity in this entity, with 49% unmutated cases (less than 2% somatic mutations). The 7q31 deletions and a shorter overall survival were more frequent in this group. Additionally a high percentage (18 of 40 sequences) of SMZL cases showed usage of the VH1-2 segment, thereby emphasizing the singularity of this neoplasia, suggesting that this tumor derives from a highly selected B-cell population and encouraging the search for specific antigens that are pathogenically relevant in the genesis or progression of this tumor.

Description: AIM/CD69 is the earliest leukocyte activation antigen and is expressed mainly by activated T, B, and natural killer (NK) cells. It is also constitutively expressed by platelets, by bone marrow myeloid precursors, and by small subsets of resident lymphocytes in the secondary lymphoid tissues. The engagement of CD69 by specific antibodies induces intracellular signals, including Ca++ flux, cytokine synthesis, and cell proliferation. To investigate the physiological relevance of CD69, we generated mice deficient in CD69 (CD69-/-) by gene targeting in embryonic stem cells. CD69 (-/-) mice showed largely normal hematopoietic cell development and normal T-cell subpopulations in thymus and periphery. Furthermore, studies of negative- and positive-thymocyte selection using a T-cell receptor transgenic model demonstrated that these processes were not altered in CD69 (-/-) mice. In addition, natural killer and cytotoxic T lymphocyte cells from CD69-deficient mice displayed cytotoxic activity similar to that of wild-type mice. Interestingly, B-cell development was affected in the absence of CD69. The B220hiIgMneg bone marrow pre-B cell compartment was augmented in CD69 (-/-) mice. In addition, the absence of CD69 led to a slight increase in immunoglobulin (Ig) G2a and IgM responses to immunization with T-dependent and T-independent antigens. Nevertheless, CD69-deficient lymphocytes had a normal proliferative response to different T-cell and B-cell stimuli. Together, these observations indicate that CD69 plays a role in B-cell development and suggest that the putative stimulatory activity of this molecule on bone marrow-derived cells may be replaced in vivo by other signal transducing receptors.