ALBERT

Beschreibung: Backround: Arsenic trioxide (ATO) has been shown to be synergistic with melphalan both in vitro and in vivo. We conducted a phase I/II trial to determine the safety and efficacy of a combination of arsenic trioxide, melphalan and ascorbic acid (AA) as preparative regimen in patients undergoing high-dose therapy (HDT) and autologous hematopoietic progenitor cell transplantation for multiple myeloma (MM). We also assessed the impact ATO levels on melphalan pharmacokinetics (PK), engraftment and toxicity. Methods: Forty-eight patients with secretory myeloma (23 females, 25 males; median age: 54, range: 3570) were treated between 4/04 and 8/05. All patient received melphalan 100 mg/m2 IV on days -4 and -3 and AA 1000 mg/day IV on days -9 to -3. Patients were randomized to 3 arms; no ATO (arm 1), ATO 0.15 mg/kg IV on days -9 to -3 (arm 2) and ATO 0.25 mg/kg IV on days -9 to -3 (arm 3). Twelve patients had disease progression or relapse after a prior autograft. Median CD34 cells dose infused was 4.5 x 106/kg (range 2.3–10.9). Results: Patients in all 3 arms were evenly matched. With a median F/U of 14.0 months (range 6–25) post autograft, no dose-limiting toxicity or non-relapse mortality was seen. Toxicity was limited to grade I or II nausea, vomiting and diarrhea. Median ATO levels on day 0 in arms 1, 2 and 3 were 0.2, 26.3 and 46.2 ng/ml, respectively. Melphalan PK was not altered by ATO pretreatment. Median time to neutrophil engraftment (ANC 〉500/ dl) was 9 days. There were no engraftment failures or delays in the ATO arms. CR rate for the entire group was 23%, and total response rate (CR + PR) was 75%. 1-year Progression-free survival (PFS) and overall survival (OS) were 75% and 95%, respectively. There was no significant difference in CR, RR, PFS or OS between the 3 arms (p = 0.9, 0.9, 0.4 and 0.6, respectively). A prior autologous transplant (p = 0.02) and abnormal cytogenetics at transplant (p = 0.04) were associated with a significantly shorter remission. Conclusions: ATO + melphalan + ascorbic acid is a safe, effective and well tolerated preparative regimen for patients with multiple myeloma undergoing an autotransplant. A longer follow up is needed to assess the impact of ATO on progression-free and overall survival.

Beschreibung: DLBCL is a curable subtype of non-Hodgkin lymphoma, although a significant number of patients do not achieve a remission or they relapse with conventional chemotherapy. While clinical variables (e.g., IPI), tumor (somatic) genetic alterations, and gene expression profiling have all been shown to predict outcome, there remains a need for additional prognostic biomarkers. One understudied class of biomarkers is host genetic background. We evaluated the hypothesis that germline variability in 73 SNPs from 44 candidate immune genes was associated with overall survival in DLBCL. We addressed this hypothesis in 365 DLBCL patients aged 20–70 years who participated in a population-based case-control study conducted from 1998–2000 (prior to the use of R-CHOP) through the Surveillance, Epidemiology, and End Results (SEER) cancer registries in Detroit, Seattle, Iowa, and Los Angeles. Germline DNA was extracted from a venous blood sample or mouthwash buccal cell sample, which was collected a median of 4.8 months after diagnosis in this population-based study. All genotyping was conducted at the National Cancer Institute Core Genotyping Facility using the Taqman platform, and was successful in over 95% of the DNA samples for the SNPs evaluated. Histology, stage, presence of B-symptoms, first course of therapy, date of last follow-up, and vital status were derived from linkage to registry databases at each study site in the spring of 2005. Cox proportional hazards analysis was used to evaluate the association between individual SNPs, adjusted for age, demographic and clinical factors. Parallel modeling strategies were used to identify the best summary multi-SNP risk score to predict survival. At a median follow-up of 56 months (range, 27-78 months) for surviving patients, there were 96 deaths in 365 patients (26%). In multivariate modeling, SNPs in IL1A (rs1800587; HRCT/TT=1.90, 1.26–2.87), IL6 (rs1800795; HRGG=1.48, 0.99–2.23), IL-10 (rs1800896; HRAG/GG=1.48, 0.91–2.38), and IFNGR2 (rs2070385; HRAG/GG=1.35, 0.86–2.11) were the strongest and most robust predictors of overall survival. A summary score of the number of deleterious genotypes from these four genes in combination with clinical and demographic variables was strongly associated with survival (p=9.3 x 10−12); Kaplan-Meier 5-year survival estimates for low, intermediate, and high risk patients were 89%, 68%, and 47% respectively. In conclusion, host genetic background as measured by germline polymorphisms in immune genes including IL1A, IL6, IL10, and IFNGR2 were associated with overall survival in DLBCL after accounting for clinical and demographic factors. These promising results require confirmation and need further evaluation in patients treated with R-CHOP in conjunction with tumor and other prognostic biomarkers.

Beschreibung: Pathological angiogenesis associated with wound healing often occurs subsequent to an inflammatory response that includes the secretion of cytokines such as tumor necrosis factor (TNF). Controversy exists on the angiogenic actions of TNF, with it being generally proangiogenic in vivo, but antiangiogenic in vitro. We find that whereas continuous administration of TNF in vitro or in vivo inhibits angiogenic sprouting, a 2- to 3-day pulse stimulates angiogenesis by inducing an endothelial “tip cell” phenotype. TNF induces the known tip cell genes platelet-derived growth factor B (PDGFB) and vascular endothelial cell growth factor receptor-2 (VEGFR2), while at the same time blocking signaling through VEGFR2, thus delaying the VEGF-driven angiogenic response. Notch signaling regulates tip cell function, and we find that TNF also induces the notch ligand jagged-1, through an NFκB-dependent mechanism. Enrichment of jagged-1 in tip cells was confirmed by immunofluorescent staining as well as by laser capture microdissection/quantitative reverse-transcription–polymerase chain reaction (qRT-PCR) of tip cells sprouting in vitro. Thus, in angiogenesis, the temporal expression of TNF is critical: it delays angiogenesis initially by blocking signaling through VEGFR2, but in addition by inducing a tip cell phenotype through an NFκB-dependent pathway, it concomitantly primes endothelial cells (ECs) for sprouting once the initial inflammatory wave has passed.

Beschreibung: A role for genetic susceptibility in non-Hodgkin lymphoma (NHL) is supported by the accumulating evidence of common genetic variations altering NHL risk. However, the pattern of NHL heritability remains poorly understood. We conducted a pooled analysis of 10 211 NHL cases and 11 905 controls from the International Lymphoma Epidemiology Consortium (InterLymph) to evaluate NHL risk among those with hematopoietic malignancies in first-degree relatives. Odds ratios (ORs) and 95% confidence intervals (CIs) of NHL and its subtypes were estimated from unconditional logistic regression models with adjustment for confounders. NHL risk was elevated for individuals who reported first-degree relatives with NHL (OR = 1.5; 95% CI = 1.2-1.9), Hodgkin lymphoma (OR = 1.6; 95% CI = 1.1-2.3), and leukemia (OR = 1.4; 95% CI = 1.2-2.7). Risk was highest among individuals who reported a brother with NHL (OR = 2.8; 95% CI = 1.6-4.8) and was consistent for all NHL subtypes evaluated. If a first-degree relative had Hodgkin lymphoma, NHL risk was highest if the relative was a parent (OR = 1.7; 95% CI = 1.0-2.9). If a first-degree relative had leukemia, NHL risk was highest among women who reported a sister with leukemia (OR = 3.0; 95% CI = 1.6-5.6). The pattern of NHL heritability appeared to be uniform across NHL subtypes, but risk patterns differed by specific hematopoietic malignancies and the sex of the relative, revealing critical clues to disease etiology.

Beschreibung: High-dose therapy plus autologous stem cell transplant (ASCT) is the standard of care for patients with multiple myeloma (MM) aged ≤65 years. Melphalan–prednisone (MP)-based therapy is the standard for non-ASCT candidates but is not typically used for transplant-eligible patients as prolonged therapy with melphalan can adversely affect stem cell collection. The phase 3 VISTA study demonstrated the superior efficacy of bortezomib plus MP (VMP) versus MP in previously untreated MM patients ineligible for ASCT. In this phase 2 study, we evaluated the efficacy of a shorter course of VMP on a different treatment schedule as induction therapy prior to ASCT or as frontline therapy in non- ASCT candidates. Patients aged ≥18 years with previously untreated MM received up to six 28-day cycles of bortezomib 1.3 mg/m2 IV, days 1, 4, 8, and 11, plus oral melphalan 6 mg/m2 and oral prednisone 60 mg/m2, days 1–7. After 2–6 cycles, ASCT-eligible patients could proceed to stem cell mobilization (G-CSF 10 mg/kg/day ± GM-CSF 250 mg/m2/ day or cyclophosphamide 4 g/m2 + GM-CSF) and conditioning with melphalan 200 mg/ m2 (140 mg/m2 if aged 〉65 years). Response was assessed every two cycles and post- ASCT by International Uniform Response Criteria. The primary end point was complete response (CR) rate to VMP. A total of 45 patients were enrolled; 27 were male. Median age was 63 years (range 33–75). MM subtype was 67% IgG, 16% IgA, and 9% each κ- and λ- light-chain; 37% of patients had ISS Stage III MM, 22% had ECOG performance status 〉1, and 70% had ≥40% plasma cells in bone marrow. In total, 20 patients proceeded to ASCT. Median duration of VMP was 4 cycles in both non-ASCT (range 1–6) and ASCT (range 2–6) patients. Response rate (best response) to VMP was 95% (42 of 44 evaluable patients), including 9% stringent CR (sCR), 9% CR (18% ≥CR [95% CI: 7%, 30%]), 27% very good partial response (VGPR), and 50% partial response (PR). Best response was achieved after cycle 2 in 10 patients, cycle 4 in 25 patients, and cycle 6 in 7 patients. All 20 ASCT patients had successful stem cell mobilization; median yield of CD34+ cells/ kg was 5.6 x 106 (range 2.3–12.2 x 106), in a median of 2 collection days. Post-transplant responses were 10% sCR, 20% CR, 55% VGPR, and 5% PR; the remaining 2 patients need further follow-up for response assessment. Response improved post-VMP to post-ASCT in 10 patients (6 PR to VGPR, 2 PR to CR, 2 VGPR to CR). After median follow-up of 14.0 months (range 7.4–47.7) and 14.6 months (range 8.2–42.9) in non-ASCT and ASCT patients, respectively, both median time to progression and progression-free survival were 19.8 months (95% CI: 14.3 months, not estimable [NE]) in non-ASCT patients and 27.9 months (95% CI: 14.6 months, NE) in ASCT patients. A total of 7 patients (5 non- ASCT, 2 ASCT) have died; 1-year survival rate was 82% (95% CI: 59%, 93%) in non- ASCT patients and 95% (95% CI: 69%, 99%) in ASCT patients. Most common grade 3/4 adverse events in all 45 patients during VMP therapy included peripheral neuropathy (24%), thrombocytopenia (20%), neutropenia (18%), and infection (9%). Only 1 patient had deep-vein thrombosis. In conclusion, VMP represents a highly effective therapy for previously untreated MM, with 45% of patients achieving VGPR or better, including 18% sCR/CR. Toxicities were predictable and generally manageable. Short-course VMP therapy did not negatively impact stem cell mobilization, supporting its use as induction therapy prior to ASCT. Very high post-transplant response rates were seen, with 85% of patients achieving ≥VGPR, including 30% sCR/CR. Since achievement of CR/VGPR is associated with improved long-term outcomes in MM, the preliminary outcome data presented here appear promising; however, longer follow-up is required.

Beschreibung: The LRF CLL4 trial randomised 777 previously untreated patients with Binet stage progressive A, B or C disease between January 1999 and October 2004 to receive either Chlorambucil, Fludarabine or Fludarabine and Cyclophosphamide. Interphase FISH for deletions of chromosome 6q, 11q, 13q, 17p and trisomy 12, IgVH gene mutational status (98% cut off), CD38 (7% cut off) and ZAP70 (10% cut off) expression were measured at randomisation on 579, 523, 535 and 478 patients respectively. Leukemic cells from 39 patients utilised the VH3-21 gene of whom 33 had homologous CDR3’s. Among the biological markers, log rank analysis showed that 〉20% p53 loss, del 11q, unmutated VH genes, high CD38 and high ZAP 70 correlated with disease progression or death (Table 1) but not deletion of chromosome 6q, 13q and trisomy 12 (p=0.7, 0.3 and 0.2 respectively). There was no difference in PFS or response duration between the 52 patients with 5–20% p53 loss and the 494 patients with no p53 loss. Multivariate Cox regression analysis showed that 〉20% p53 loss (p

Beschreibung: Background: Under conditions of iron overload, ascorbic acid is oxidised at an increased rate leading to a risk of vitamin C deficiency. With deferoxamine (DFO) standard therapy, vitamin C is usually given at a dose of 2–3mg/kg on the days of DFO infusion as this increases iron excretion by up to 30%. With deferarisox (DFX) chelation treatment, although supplementation is permitted, there is currently no information about the effects of vitamin C supplementation on iron excretion and it is often left to patients or their clinician’s discretion as to whether supplementation is given. With long-term treatment, in the absence of supplementation there is a potential risk that vitamin C deficiency will develop and this could influence response to treatment. Patients and Methods: We have measured fasting plasma vitamin C in 41 patients who have been on long term deferasirox treatment for transfusional iron overload for between 1.5 and 5 years. 32 of these patients had received no supplementation and 9 patients had received 2–3 mg/kg/ day of supplementation. We have examined whether trends in serum ferritin, myocardial T2* and liver iron, during the final year of observation, relate to plasma levels of vitamin C. Results: Fasting plasma Vitamin C was significantly lower in the 41 patients (mean=30.3μmol/l, SD=20.8) than healthy control patients (mean=60.29μmol/l SD=12.6) (P

Beschreibung: BACKGROUND: With new chelation regimes such as deferasirox, there has been interest in their effects on serum creatinine, as about one third of patients show a small non-progressive increase within a few weeks of starting treatment. A question arises as to whether creatinine increases occur with other modalities of chelation therapy. The effects of deferoxamine (DFO) on serum creatinine have not been widely studied, particularly when intensive chelation regimes are used with 24 hour (h) exposure. Intensified 24h DFO for patients with high risk iron overload has been in use for selected patients for over 20 years. However, whilst factors leading to retinal and ototoxicity are well described, effects on serum creatinine are largely confined to case reports. We postulated that small increments in serum creatinine might be an inevitable effect of treatment intensification. We therefore undertook retrospectively to examine the effects on serum creatinine in patients in whom standard DFO therapy (40mg/kg as 8–10h infusions 5 days/ week) was switched to an intensive DFO monotherapy regime with 24h/day therapy. PATIENTS AND METHODS: We examined the records of 10 patients with transfusion dependent thalassaemia attending the haematology department at University College London Hospitals, between 1989 and 2008, who required an intensification of their DFO regime as a result of failure to control their iron burden with their existing regime. Nine out of the 10 patients required portacath insertion for their DFO to be delivered intravenously, the tenth patient was switched to subcutaneous DFO administered 24h/ day. The patient characteristics were as follows: 8 male and 2 female. Two had thalassaemia intermedia and the rest had thalassaemia major. Those with a portacath were commenced on warfarin as thromboprophylaxis for the portacath. Co-morbidities for the patients included splenectomy (2); diabetes (2); cardiac failure (3); arrhythmias (2). The mean dose of deferoxamine administered was 52mg/kg/24h (9 of the patients had doses between 35–60 mg/kg/24h and only 1 patient had a dose of 100 mg/kg/24h). RESULTS: The results of the study revealed a mean ferritin of 6113.2μg/L ± 1681.3 and 3910.5μg/L ± 1438.4 pre and post intensification of DFO respectively. Of the 10 patients, 6 of them displayed an increase in creatinine of over 25% of their original level on standard DFO, the other 4 also had an increase albeit a more modest one. The increase in creatinine did not correlate with the dose of DFO given. The creatinine pre-intensification showed a mean of 62.3μmol/L ± 10.19 and post intensification showed 92.3μmol/L ± 11.17. This was a statistically significant difference with a p value of 0.0016 using a paired student t test. No patient went on to develop progressive renal dysfunction and creatinine returned to baseline in all patients when the regime returned to standard therapy. CONCLUSIONS: We conclude that intensification of DFO using 24h chelation results in small increments in serum creatinine, even when doses are as low as 40–70mg/kg. The findings suggest that increments in creatinine with chelation therapy are more frequent than hitherto recognised and may be a general effect of continuous chelation therapy. These changes were reversible on returning to original intermittent DFO. While none of these patients developed permanent changes, we propose that patients who are commenced on DFO, especially at continuous intensified doses, should have their creatinine measured on a regular basis, and caution should be employed with those patients who have borderline high creatinine.

Beschreibung: Chronic iron overload associated with hereditary hemochromatosis or repeated red cell transfusions is known to cause cardiac failure. Cardiac arrhythmias have been incidentally noted in patients with iron overload, but often times dismissed as being caused by other co-morbid conditions. Studies with iron-loaded gerbils suggest a role for iron in the development of cardiac arrhythmias, however these studies utilized short duration recordings of anesthetized gerbils. Furthermore, we were unable to reproduce these loading protocols without significant morbidity and mortality. Our goal was to characterize iron-induced arrhythmias in the chronically instrumented, untethered, telemetered gerbil. Monitored gerbils were divided into 2 groups: iron-loaded (n=23) and control (n=8). Iron loaded gerbils received iron dextran intraperitoneally at a dose of 1.7 (n=4), 3.0 (n=5) or 6.2 (n=14) g/kg; control gerbils received dextran. Gerbils were weighed and given a physical exam weekly. Electrocardiograms were recorded for 10 seconds every 30 minutes for approximately 6 months (DSI Ponehma) and reviewed daily. Quantitative analysis was completed on 6 iron loaded (6.2g/kg) and 3 control gerbils. Heart rate and intervals were calculated and arrhythmias were characterized and counted. Cardiac and hepatic histology and tissue iron concentration were assessed. All gerbils showed evidence of frequent sinus arrhythmia (more than one episode per hour). However, except for two control gerbils that showed frequent unifocal PVCs, no significant arrhythmias were noted in daily review. There was no difference in heart rate, P duration, PR interval, QRS duration or QT interval between groups. Neither total number of arrhythmias nor arrhythmias per minute were different between groups. One iron-loaded gerbil had a single episode (11 beats) of supraventricular tachycardia. Two iron-loaded gerbils had PVCs, one had only a single beat and the other had 9 unifocal PVCs over the duration of the study. Iron-loaded gerbils rarely showed other arrhythmias One control gerbil had 260 unifocal PVCs over the duration of the study. Other arrhythmias were noted rarely. Body weight and heart weight was not different between groups, while liver weight increased with increasing iron dose. Cardiac and hepatic iron were significantly increased in iron loaded gerbils when compared to control. Liver weight increased as iron dose increased. Seven of 14 gerbils loaded to 6.2 g/kg developed ascites as assessed both by physical examination and necropsy. We conclude that an iron load sufficient to cause clinical liver disease does not, in the absence of co-morbid conditions, cause cardiac arrhythmias in the gerbil model of iron overload. This suggests that iron alone is insufficient to cause cardiac arrhythmias.

Beschreibung: Background: Leukemia is the most common pediatric malignancy, accounting for nearly 40% of all new childhood cancer. The cure rates for pediatric ALL have increased to more than 80% and the cure rates for pediatric AML now approach 50%. Much of this progress can be attributed to cytogenetic and molecular risk stratification with subsequent randomized control trials. Genomic instability events may also serve as relevant prognostic biomarkers. A novel high-throughput genomic technology called Molecular Inversion Probes (MIPs) quantifies genomic instability, gene copy number and allelic imbalances at the highest genomic resolution. MIPs can analyze genetic target sequences in parallel with high specificity and sensitivity. Further classifying leukemic blasts using more precise molecular techniques could prove useful in further risk stratifying and developing new treatment strategies. Objective: To adapt MIPs to characterize and define unique molecular subtypes of childhood leukemia. Methods: DNA was extracted from AML and ALL clinical samples obtained at diagnosis. 400 ng of leukemic DNA was mixed with a previously synthesized MIP cancer probe set (667 probes representing all human chromosomes from exon sequences of 298 cancer genes); each probe contains two unique recognition sequences to targeted genomic DNA and a unique barcode (tag) sequence. A thermostable polymerase and ligase were added, and the mixture was denatured. Probe homology sequences were then annealed to complementary genome sites. Standard PCR reagants were added including one fluorescent-labeled primer and unlabeled primer. The reaction was thermocycled and probes circularized in the nucleotide specific “gap fill” reaction were amplified. The PCR mixture was hybridized to a barcode microarray overnight. The chip was stained and washed, and then interrogated by an Affymetrix scanner via argon laser excitation at 488-nm. ‘Cluster along Chromosomes’ analysis was performed to detect significant amplifications or deletions. Results: Childhood ALL and AML samples had unique patterns of multiple gene deletions. Additionally, overlap was found in several of these deleted genes. Significantly deleted genes in common between ALL and AML samples included: RAP1GA1, HYAL1, HSPA9B, CSF1R, TNF, NOTCH4, FANCE, CCND3, BAG2, PLAGL1, MAD1L1, POLM, POLD2, IGFBP3, PSD, SUFU, CYP17A1, MXI1, HRAS, IGF2, RRM1, BRCA2, TP53, ERBB2, ERCC2, and POLD1. Conclusions: MIPs represent a novel technology for highly sensitive and specific gene copy analysis of childhood leukemia. Unique and distinguishing signatures of allelic imbalance can be determined between ALL and AML clinical samples using MIP technology. The unexpected overlap of deleted genes in both ALL and AML may represent a common molecular mechanism that requires further investigation. We currently are adapting MIPs for an expanded cancer probe set and are exploring new techniques to determine loss of heterozygosity (LOH) in pediatric leukemia.