ALBERT

Description: Introduction: The human parvovirus B19 (pvB19) is well known as a cause of chronic anemia and red cell aplasia, this due to the high tropism for the erythroid progenitor cells where it replicates. Some reports have suggested that the infection of this virus is related to an erythropoietin resistance in patients with chronic anemia and kidney failure. The most frequent causes of erythropoietin resistance are iron deficiency, chronic inflammation process, vitamin deficiency, hyperparathyroidism and malignancies among others. The aim of this study was to detect the viral DNA of PvB19 and its correlation with hemoglobin levels and human erythropoietin dosage in patients with kidney failure under hemodialysis treatment. Patients and Methods: All patients had chronic renal failure and were undergoing hemodialysis. The detection of the viral DNA was performed by qualitative polymerase chain reaction (PCR). The PCR reaction was carried out using one specifically designed primer set for PvB19 that encoded for a structural capsid protein (VP1). Erythropoietin resistance (ER) was diagnosed using the European Best practice Guidelines as stated by Hay N (Nephrology, 2002). Results: A total of 58 samples of peripheral blood were obtained from patients with chronic kidney failure. Mean age was 59.41 (23 to 85 years) and mean Hemoglobin levels were 10.1 g/dL (7–14). Iron was normal in all patients and no patient had active infections. Fifty two patients (89.65%) were receiving recombinant erythropoietin. Among all the samples, 3 cases met ER criteria but only in 1 of them did we find the viral DNA (1.72%). Three cases (5.17%) positive to the viral DNA were found in the non erythropoietin resistance group. Conclusion: Although ours is a small group and we are still gathering samples, up until now, no relation appears to exist between the presence of PvB19 genetic material and ER.

Description: Waldenström’s macroglobulinemia (WM) is an incurable low-grade lymphoproliferative disorder characterized by bone marrow (BM) infiltration of a clonal population of small B-lymphocytes, plasmacytoid lymphocytes and plasma cells that secrete monoclonal IgM antibody. Because it is difficult to obtain tumor metaphases for karyotype studies, few recurrent chromosomal abnormalities have been reported and the genetic basis of the disease remains poorly defined. Therefore, we performed a comprehensive high-resolution study to identify genomic abnormalities involved in WM pathogenesis. Fifty-seven WM patients were analyzed using a combination of array-based comparative genomic hybridization (aCGH)(N=42); gene expression profiling (GEP)(N=22), DNA sequencing (N=24) and cIgM-FISH (N=57). Agilent 244A and Affymetrix U133 platforms were used in the aCGH and GEP experiments, respectively. An NF-κB index was calculated from GEP data, as a surrogate marker of NF-κB transcription activity. NFKB2 sub cellular localization was determined by immunofluorescence staining. Overall, 83% of samples have aCGH defined chromosomal abnormalities, with a median of 3 abnormalities per patient (range 0 to 27). We identified 16 recurrent regions of copy number change found in 〉5% (3 or more patients); 10 deleted and 6 amplified regions. The most common abnormality was the entire or partial deletion of 6q, identified in 40% of cases. Four nonoverlapped minimal deleted regions were identified on 6q (MDR1 to MDR4), each of them being present in at least 14 of 17 patients with the abnormality. Gain of 6p was the second most common abnormality (17%) and its presence was always concomitant with 6q loss. Other recurrent deletions were 13q14 (10%) and 7q22, 8p, 11q22-q23, 11q23-q24 and 17p11-p13 (7% each). Copy gains were identified as partial or entire gains of chromosomes 18 (17%), 4 (12%) and 3 (10%), 8q (10%) and Xq27.1-q28 (10%). Three patients had either homozygous deletions or LOH with mutations affecting both alleles of TRAF3 on 14q32. TRAF3 inactivation was correlated with an increased NF-kB transcriptional signature and it was associated with activation of the non-canonical NFkB pathway. Additionally, we identified the inactivation of TNFAIP3, another negative regulator of the NF-kB pathways, in one of 24 patients. Finally, interstitial deletions identified in 4 patients at 13q14 identified a 1.1 Mb MDR, including MIRN15A and MIRN16-1. These findings confirm that focal 13q14 deletion is not restrict to B-CLL and that the MIRN15A/MIRN16-1 abnormalities might be common to several indolent B-cell diseases. Overall, we identified TRAF3 and TNFAIP3 inactivation in 5.3% and 2.4%, respectively. To our knowledge, this is the first study showing the inactivation of bona fide tumor suppressor genes in WM patients. Furthermore, these genes are negative regulators of NF-kB signaling pathway, highlighting its biologic importance in sustaining and limiting growth in WM. Mutational activation of this pathway, which is normally activated by ligand-receptor interactions within the BM microenvironment, suggest a therapeutic role for inhibitors of NF-KB pathway activation in the treatment of WM.

Description: Lenalidomide is active in lymphoid malignancies, but its mechanism of action remains ill defined. One possible mechanism is immune activation due to increased expression of costimulatory molecules on tumor cells. In CLL lenalidomide treatment has been uniquely complicated by tumor flare reactions (TFR: pain and lymph node swelling) resulting in treatment related mortality. To investigate effects of lenalidomide on CLL cells we exposed PBMC from 17 CLL patients enrolled in a phase II clinical trial of single agent lenalidomide and normal donors (n=10) in-vitro to 2μM lenalidomide for 48 hours and measured costimulatory molecules CD80 and CD86 on B-cells and activation marker CD69 on T-cells by flow cytometry. CD80 expression increased on average 2-fold on CLL cells but remained unchanged on normal B-cells (p=0.01 for log2 MFI CLL vs normal). CD69 expression on T-cells followed a similar pattern, albeit with more interindividual variability among CLL samples (p=0.03 for log2 MFI CLL vs. normal). Next we wished to correlate the degree of in-vitro activation with the clinical effects of lenalidomide treatment in the same patient. Our observations in several patients suggested that the dominant feature of lenalidomide treatment is a cytokine release syndrome (CRS). Indeed, on day 8 of treatment we detected increased serum levels of TNFa, IL-1ra, CCL2, CCL3, CCL4 and IL-8. To correlate the CRS with in-vitro measurements, we applied uniform criteria for diagnosis. Patients who experienced at least 2 of the following symptoms (% of patients with the symptom, n=18) were considered to have a CRS: increase in lymph node size and or ALC by 〉25% (50%), fever 〉38C (44%), pain (61%), fatigue (72%), chills (33%), hypotension/dehydration (39%), and rise of creatinine (33%). Onset of symptoms was within 8 to 72 hours (average 38 hours) after initiation of therapy (20mg patients 1–10, 10mg patients 11–18). The CRS score, summarizing number and severity of symptoms in each patient, averaged 3.14 (range 0–10) with no difference between the 20mg and10mg cohort. The CRS score correlated (Pearson r-value, p50% in CD3+ cells in the lymph node and the average pre/post ratio of T-cells was 1.14 (p=0.37). In summary, lenalidomide upregulated expression of CD80 on B-cells and of CD69 on T-cells from CLL patients but not on normal B and T-cells. The in-vitro response correlated with the clinical onset and severity of a CRS in-vivo. However, the degree of T-cell activation or of the CRS did not predict the effect on the leukemic cell count. Our data suggest the possibility that immune activation in CLL may be primarily responsible for side effects but not be required for disease control. This interpretation is consistent with the good clinical activity of lenalidomide in several lymphoid malignancies in the absence of notable immune effects and if confirmed, has important implications for future use of lenalidomide and the formulation of combination regimens.

Description: Centrosome amplification is common in myeloma and may be involved in disease pathogenesis. We have previously derived a gene expression–based centrosome index (CI) that correlated with centrosome amplification and was an independent prognostic factor in a small cohort of heterogeneously treated patients. In this study, we validated the prognostic significance of the CI in 2 large cohorts of patients entered into clinical trials and showed that a high CI is a powerful independent prognostic factor in both newly diagnosed and relapsed patients, whether treated by intensive therapy (total therapy II) or novel agents (bortezomib). Tumors with high CI overexpressed genes coding for proteins involved in cell cycle, proliferation, DNA damage, and G2-M checkpoints, and associated with the centrosome and kinetochore/ microtubules. In particular, aurora kinases are significantly overexpressed in patients with high CI, with concordant increase in protein expression. Human myeloma cell lines with higher CI are more responsive to treatment with a novel aurora kinase inhibitor. Aurora kinase may represent novel therapeutic targets in these patients with very poor prognosis.

Description: Abstract 5096 Background Deferasirox is a once-daily oral iron chelator with established dose-dependent efficacy for treating transfusional iron overload. The retrospective multicenter Brazilian trial included patients (pts) from 14 sites of 10 states with a variety of transfusion-dependent anemias and was designed to evaluate the efficacy and safety of fixed starting doses of deferasirox based on transfusion history, with subsequent dose titration based on serum ferritin (SF) trends. Data were available from 105 eligible pts at 6 months of treatment and 73 pts from 1-year follow-up period. Methods Pts (aged ≥ 2 yrs) had transfusion-dependent anemia with a history of multiple transfusions (〉20 transfusions) and/or SF levels ≥ 1000 ng/mL and serum creatinine level 〈 the upper limit of normal (ULN). Deferasirox starting dose was 10-30mg/kg/day depending on transfusion requirements and subsequent dose adjustments of 5-10 mg/Kg/day (range 0–35 mg/kg/d) were done every 3 months based on changes in SF and safety parameters. Efficacy was assessed monthly by measuring change from baseline in SF levels. Safety was evaluated on a monthly basis according to the incidence and type of adverse events and measurement of laboratory parameters, including serum creatinine and liver enzyme levels. Results 105 pts (40 M, 65 F; mean age 25.0±16.6 yrs) were enrolled; 46% (n=48) aged 40 units of red blood cell (RBC); 71.5% (n=75) were on regular RBC transfusion, 56% of the pts required 〈 2 RBC units/month and 44% between 2 and 4 RBC units/month. Only 63% (n=66) had received prior chelation therapy: deferoxamine (DFO; 51.4%) or DFO/deferiprone combination (11.4%). Sixty-four (61%) pts started on 20 mg/kg/d and 41(39%) 〉 20-30 mg/kg/d, 15.2% of pts had dose increases at a median of 24 weeks after treatment initiation. Mean ± SD SF levels (μg/L) did significantly reduce at 6 months and 12 months compared to baseline (BL) [from 3132.14 ± 2237.47 to 2784.25 ± 1969.7 at 6 months (p=0.0001) and 2327.46 ± 1873.8 at 12 months (p=0.005)]. The proportion of patients with SF levels 〈 2000, 2000-3000 and 〉 3000 μg/L from BL to 6 and 12 months by percentage of patients changed from 36% to 47.5% and 52%; from 26% to 26.5% and 24.5%; from 38% to 26% and 23%, respectively. No patient discontinued the treatment. No death was reported by the investigators during the study. The most common drug-related (investigator-assessed) AEs were mild, transient diarrhea (n=15; 14.3%), rash (n=5; 4.7%), nausea (n=9; 8.5%) and headache (n=6; 5.7%). Seven pts (6.6%) had serum creatinine value 〉33% above BL on two consecutive visits, 3 (2.8%) of whom had creatinine increases above the ULN; there were no progressive increases or renal failure. Eleven (10.5%) pts had an increase in alanine aminotransferase 〈 5x ULN but no one experience increases ≥ 5x ULN; levels were already elevated in all of them. Conclusions This first multicenter Brazilian study confirms deferasirox efficacy in achieving a reduction of iron load across a wide range of pts with transfusion-related iron overload. It also supports the clinical approach to fixed starting dose of deferasirox based on iron intake from ongoing blood transfusions and current iron burden with subsequent individual dose titration every 3 months according to SF trends and safety markers. Deferasirox was generally well tolerated in pediatric and adult pts with a safety profile consistent with data from previous clinical trials. The availability of deferasirox as a once-daily oral iron chelator would potentially facilitate improved compliance, and thereby reduce morbidity and mortality from iron overload. Disclosures No relevant conflicts of interest to declare.

Description: Abstract 3427 Poster Board III-315 Lenalidomide (L) trials in relapsed chronic lymphocytic leukemia (CLL) have shown promising results when the drug was given continuously or with short interruptions. Here we present data from a phase II clinical trial (ClinicalTrials.gov Identifier: NCT00465127) in which L was given for 3 weeks followed by a 3 week drug-free period. This dosing scheme was devised based on the hypothesis that immune stimulatory effects might be more effective when L is pulsed and to determine whether an extended recovery period would reduce toxicities, especially myelosuppression. This single center, investigator initiated trial, enrolled patients with CLL or small lymphocytic lymphoma, who had relapsed after standard of care therapy, had a neutrophil count 〉500/ul and a platelet count 〉20,000/ul. The primary endpoint of the study was response after 4 cycles of L. Patients with at least a partial response were allowed to receive up to 4 additional cycles. The starting dose for the first 10 patients was 20 mg daily; it was then lowered to 10 mg daily because of toxicities observed in other L trials for CLL. A dosage increase was allowed after cycle 2 if blood counts permitted. TLS prophylaxis with allopurinol was mandated during cycle 1-3. Deep venous thrombosis (DVT) prophylaxis was not mandated unless risk factors were present. Ibuprofen and corticosteroids were allowed to treat inflammatory symptoms. The planed accrual goal has been reached (27 patients). Patient characteristics were: median age 64 years (36-78), median number of prior therapies was 3 (range 1-7), 14 patients (52%) were in Rai stage III-IV, 14 patients (52%) had del 17p, 18 patients (67%) had bulky disease, 12 out of 22 patients (55%) were ZAP70+, and in 13 of 18 patients (72%) the CLL clone expressed unmutated immune globulin VH genes (〉98% germline). 26 patients received at least 1 cycle of therapy (range 2-8) and could be evaluated for toxicity, 1 patient never started treatment. Toxicities included Gr 3/4 neutropenia in 20 patients (77%) that worsened with cumulative cycles, 3 patients (12%) developed febrile neutropenia and G-CSF was given to 20 patients (77%), mostly in later cycles; Gr 3/4 thombocytopenia in 8 patients (31%); and DVTs (Gr 3) in 5 patients (19%). A cytokine release syndrome (CRS), that includes reactions labeled as tumor flare syndrome, was observed in 19 patients (73%) in cycle 1, including Gr 3/4 reactions in 5 patients (19%). The CRS recurred in 11 (42%), 5 (19%) and 3 patients (12%) in cycles 2, 3, and 4 respectively. No case of tumor lysis syndrome was observed. Non-neutropenic infections were seen in 13 patients (50%), mainly Gr 1/2, but 1 patient died from S. pneumoniae sepsis at the end of cycle 4. CD4 lymphopenia Gr 3/4 occurred in 9 patients (35%) and one patient with Gr 4 developed CMV colitis. 18 patients received at least 4 cycles of L and were evaluable for response: 6 (33%) had partial response (PR), 9 (50%) stable disease, and 3 (17%) progressive disease. 5 of 6 patients with PR (84%) had a del 17p and bulky disease. Once treatment was stopped, duration of response was short (median 3.5 months, range 1-13). 6 patients (26%) could not complete 4 cycles of L; 2 because of autoimmune complications, 3 because of side effects, and 1 because of PD in cycle 3. In summary, L cycled 3 weeks on, 3 weeks off induced responses, all partial, in 33% of relapsed CLL patients, comparable to previous studies using other dosing strategies. We observed remarkably good activity in patients with 17p deletion and bulky nodal disease. Unfortunately, pulse dosing of L did not lead to reduced toxicities and myelosuppression remained a major concern and was worsening with cumulative cycles. Notable additional toxicities of L in CLL indentified in this study included CD4 lymphopenia, in one case complicated by CMV colitis, and a 19% incidence of DVTs. Once L was discontinued, the duration of response was short, suggesting a need for continued therapy in patients who are able to tolerate the drug. Disclosures Off Label Use: Lenalidomide is not FDA approved for chronic lymphocytic leukemia.

Description: Busulfan(Bu)-based chemotherapy is a conditioning treatment prior to hematopoietic stem cell transplantation (HSCT) of patients with acute and chronic myelogenous leukemia (AML, CML). A major obstacle to successful HSCT is Bu resistance, which might be attributed to individual differences in drug pharmacokinetics and metabolism, or inherent resistance of cancer cells. We hypothesize that gene expression profiling of leukemia cells can be used to dissect the factors that contribute to their Bu resistance. Two Bu-resistant leukemia cell lines were established, characterized and analyzed by microarray and real-time RT-PCR techniques to identify differentially expressed genes. The CML B5/Bu2506 cells are 4.5-fold more resistant to Bu than their parental B5 cells. The AML KBM3/Bu2506 cells are 4.0-fold more Bu-resistant than KBM3 parental cells. Both resistant sublines evade Bu-mediated G2-arrest and apoptosis with constitutively up-regulated anti-apoptotic genes (BCL-XL, BCL2, BCL2L10, BAG3 and IAP2/BIRC3) and down-regulated pro-apoptotic genes (BIK, BNIP3, and LTBR).). Bu-induced apoptosis is partly mediated by activation of caspases; use of the inhibitor Z-VAD-FMK completely abrogated PARP1 cleavage and reduced apoptosis by ∼ 50%. Furthermore, Bu resistance in these cells may be attributed in part to up-regulation of HSP90 protein and activation of STAT3. Inhibition of HSP90 with geldanamycin attenuated phosphorylated STAT3 and made B5/Bu2506 and KBM3/Bu2506 more Bu-sensitive. Analysis of cells derived from patients classified as either clinically resistant or sensitive to high-dose Bu-based chemotherapy had alterations in gene expression that were analogous to those observed in the in-vitro model cell lines, confirming the potential clinical relevance of this model for Bu resistance. Our results suggest the important roles of apoptotic signaling mechanism, HSP90 and STAT3 and should be considered in the classification of AML patients who will likely benefit from busulfan-based pretransplant conditioning therapy and those who should be offered alternative treatment modalities.

Description: The DNA-alkylating drug busulfan (Bu) is commonly used in myeloablative pretransplant conditioning therapy in patients with chronic myelogenous leukemia (CML). A major obstacle to successful treatment is inherent or acquired cellular Bu-resistance. We hypothesized that cellular Bu-resistance can be reversed by epigenetic upregulation of pro-apoptotic genes. We established a Bu-resistant CML cell line B5/Bu2506 which is 4-fold more resistant to Bu than the parental B5 cells but 4-fold collaterally more sensitive to DNA demethylating agent 5-aza-2′-deoxycytidine (DAC). Exposure to DAC synergistically increased Bu-mediated cytotoxicity in B5/Bu2506 cells as evaluated by the MTT assay and analyzed by the median-effect method (combination index CI 〈 0.5). The DAC-induced sensitivity to Bu of B5/Bu2506 cells was associated with PARP1 cleavage, phosphorylation of histone 2AX and activation of caspase 8. Real-time PCR and immunostaining analyses of the expressions of various pro-apoptotic genes, which are known to be epigenetically regulated, showed a highly significant increase (approximately 40-fold) in the expression of XAF1 (X-linked inhibitor of apoptosis protein (XIAP)-associated factor 1) in B5/Bu2506 cells that were exposed to 0.5 microM DAC for 48 hrs. Analysis of the XAF1 promoter by methylation-specific PCR showed a significant decrease in its methylation status which correlates with the alteration in its gene expression in the presence of DAC. These findings will be discussed relative to our analysis of XAF1 gene methylation status in leukemia cell samples obtained from patients who underwent allogeneic stem cell transplantation after high dose Bu-based conditioning therapy and either remained in remission for more than one year or who experienced rapidly progressive leukemia. Our results suggest that DNA methylation status (eg. XAF1 gene) may be used to identify leukemia patients who could benefit from Bu-DAC combinations to achieve improved leukemic cytoreduction with the conditioning program.

Description: Germ-line mutations in bone morphogenic protein type II receptor (Bmpr2) confer susceptibility to pulmonary arterial hypertension (PAH), which is characterized by obstructive vascular lesions in small arteries. The molecular and cellular mechanisms that account for the etiology of this disorder remain elusive, as does the role of Bmpr2 in postnatal tissue homeostasis. Here we show that in adult mice, stably silencing Bmpr2 expression by RNA interference does not increase pulmonary arterial resistance but results in severe mucosal hemorrhage, incomplete mural cell coverage on vessel walls, and gastrointestinal hyperplasia. We present evidence that BMP receptor signaling regulates vascular remodeling during angiogenesis by maintaining the expression of endothelial guidance molecules that promote vessel patterning and maturation and by counteracting growth factor–induced AKT activation. Attenuation of this function may cause vascular dysmorphogenesis and predisposition to angioproliferative diseases. Our findings provide a mechanistic link between PAH and other diseases associated with the BMP/TGF-β pathways, such as hereditary hemorrhagic telangiectasia and juvenile polyposis syndrome.