ALBERT

Description: Fabry disease is an X-linked disorder associated with early-onset stroke, cardiomiopathy, and progression to end-stage renal failure. Correlations between IL-6 inflammatory cytokine gene polymorphisms and Mainz Severity Score Index (MSSI) scores have been shown. Therefore, polymorphisms of other key pro- and anti-inflammatory cytokines and correlation to clinical manifestations (MSSI scores) were attempted in order to build haplotype descriptions for Fabry disease. Genotyping for IL-10[819C/T;-592C/A]; IL-1b[+3954 C/T; -511C/T]; IL-1α[-889C/T]; and TNF-α[–308G/A] was performed in 76 patients and correlated with MSSI sub-scores and with enzyme (alpha-galactosidase A) levels. Fifty normal volunteers, age- and sex-matched, were also genotyped. Of 76 patients, 31(41%) were males and 45 (59%) were females. There was no correlation between enzyme levels and any cytokine levels. Statistically significant differences were found in prevalence of TNF-α [–308G/A] genotypes: 84%GG in patients versus 63%GG in controls (p=0.038) and for IL–1α [–889C/T] genotypes: 94%CC in patients versus 21%CC in controls (p

Description: High levels of granulocyte/macrophage–colony-stimulating factor (GM-CSF) autoantibodies are thought to cause pulmonary alveolar proteinosis (PAP), a rare syndrome characterized by myeloid dysfunction resulting in pulmonary surfactant accumulation and respiratory failure. Paradoxically, GM-CSF autoantibodies have been reported to occur rarely in healthy people and routinely in pharmaceutical intravenous immunoglobulin (IVIG) purified from serum pooled from healthy subjects. These findings suggest that either GM-CSF autoantibodies are normally present in healthy people at low levels that are difficult to detect or that serum pooled for IVIG purification may include asymptomatic persons with high levels of GM-CSF autoantibodies. Using several experimental approaches, GM-CSF autoantibodies were detected in all healthy subjects evaluated (n = 72) at low levels sufficient to rheostatically regulate multiple myeloid functions. Serum GM-CSF was more abundant than previously reported, but more than 99% was bound and neutralized by GM-CSF autoantibody. The critical threshold of GM-CSF autoantibodies associated with the development of PAP was determined. Results demonstrate that free serum GM-CSF is tightly maintained at low levels, identify a novel potential mechanism of innate immune regulation, help define the therapeutic window for potential clinical use of GM-CSF autoantibodies to treat inflammatory and autoimmune diseases, and have implications for the pathogenesis of PAP.

Description: Purpose/Objective: To evaluate patterns of recurrence in patients with Hodgkin’s (HL) and non-Hodgkin’s lymphoma (NHL) who subsequently undergo autologous stem cell transplant (ASCT). In this population that has declared itself as high risk, we evaluated time to and sites of relapse relative to initial sites of disease and radiation therapy (RT). This information might enhance understanding of the natural history of these diseases in the setting of modern therapy, influence treatment strategies, and assist in screening decisions. Materials/Method: We analyzed the records of all 281 consecutive patients with refractory or recurrent HL and NHL (indolent and aggressive, as defined at initial diagnosis) who underwent ASCT in our center between 5/92–7/03. Patients were initially diagnosed between 1979–2003 at a median age of 44 years (8–70). 25 patients were unevaluable due to insufficient data, and 68 patients were excluded from analysis because their disease was refractory to initial and salvage therapy. HL patients were segregated according to initial staging (I/II vs. III/IV). Results: Early stage HL patients relapsed at a median of 2.0 years (0.5–10.3) with 87% relapsing in initial disease site(s); 13% (95% CI 3.8–30.1%) relapsed only in new sites. Advanced stage HL patients relapsed at a median of 1.4 years (0.6–10.5) with 96% relapsing in initial site(s); 4% (95% CI 0.1–21.9%) relapsed only in new sites. Indolent histology NHL patients relapsed at a median of 2.1 years (0.5–14.9) with 83% relapsing in initial site(s); 17% (95% CI 7.3–32.8%) relapsed only in new sites. Aggressive histology NHL patients relapsed at a median of 1.0 year (0.3–8.0) with 64% relapsing in initial site(s); 36% (95% CI 26.2–46.2%) relapsed only in new sites. For early stage HD patients, recurrences were predominantly local, and uniformly so in those unirradiated. For all other groups, fewer patients were irradiated than unirradiated and local recurrences predominated regardless of therapy. Conclusions: Almost all patients with HL who relapse and subsequently undergo ASCT initially recur in previous disease sites. Although patients with aggressive histology NHL are more likely to relapse in new sites than patients with indolent NHL, local recurrences predominate in both groups. The median time to recurrence is brief (1–2.1 years). In a population defined by recurrent disease, it is expected that relapses will occur in irradiated sites. Relative protection by RT of local recurrence cannot be determined until all patients, regardless of relapse status, are analyzed. However, these data support an emphasis on local control and suggest that the frequency of screening be most rapid in the early post-therapy years. Comparison of Site(s) of Relapse to Site(s) of Initial Presentation HL NHL Early (n=30) Adv. (n=23) Ind. (n=40) Agg. (n=95) Characteristic % % % % New Site(s) 13 4 18 36 Previous site(s) only 63 61 60 44 Previous site(s) + new site(s) 23 35 23 20 Characteristic n n n n Radiated patients relapsing in previous site(s) 15/19 6/6 5/7 20/34 Unradiated patients relapsing in previous site(s) 11/11 16/17 26/33 42/61

Description: The trial ALL-BFM 95 for treatment of childhood acute lymphoblastic leukemia was designed to reduce acute and long-term toxicity in selected patient groups with favorable prognosis and to improve outcome in poor-risk groups by treatment intensification. These aims were pursued through a stratification strategy using white blood cell count, age, immunophenotype, treatment response, and unfavorable genetic aberrations providing an excellent discrimination of risk groups. Estimated 6-year event-free survival (6y-pEFS) for all 2169 patients was 79.6% (± 0.9%). The large standard-risk (SR) group (35% of patients) achieved an excellent 6y-EFS of 89.5% (± 1.1%) despite significant reduction of anthracyclines. In the medium-risk (MR) group (53% of patients), 6y-pEFS was 79.7% (± 1.2%); no improvement was accomplished by the randomized use of additional intermediate-dose cytarabine after consolidation. Omission of preventive cranial irradiation in non–T-ALL MR patients was possible without significant reduction of EFS, although the incidence of central nervous system relapses increased. In the high-risk (HR) group (12% of patients), intensification of consolidation/reinduction treatment led to considerable improvement over the previous ALL-BFM trials yielding a 6y-pEFS of 49.2% (± 3.2%). Compared without previous trial ALL-BFM 90, consistently favorable results in non-HR patients were achieved with significant treatment reduction in the majority of these patients.

Description: Invasive aspergillosis remains a serious complication in patients undergoing allogeneic stem cell transplantation (SCT). Since it became clear that lymphocytes provide a critical secondary defense against fungi, adoptive transfer of functionally active anti-Aspergillus T cells might be an option to restore adaptive immune effector mechanisms. Using the interferon (IFN)-γ secretion assay, we isolated human activated T cells upon stimulation with a cellular extract of Aspergillus fumigatus. Culturing this cell population for 14 days, we obtained an average of 1.1 × 107 cells from a single 100-mL blood draw in 7 of 7 healthy individuals. Within another 14 days, these cells were expanded to an average number of 2.0 × 108 T-helper 1 (TH1) cells secreting IFN-γ on stimulation with Aspergillus antigens. Testing various fungal antigen extracts, similar proportions of IFN-γ-producing CD3+/CD4+ cells were obtained upon activation with antigen extracts of A fumigatus, A flavus, A niger, and Penicillium chrysogenum, whereas no significant IFN-γ production was observed upon activation with antigen extracts of Alternaria alternata and Candida albicans. In addition, generated T cells were able to induce damage to A fumigatus hyphae, and significantly increased hyphal damage induced by human neutrophils. CD4+ T-cell-mediated alloreactivity of generated anti-Aspergillus T cells was clearly reduced compared with that of the original cell population. In conclusion, we present a simple and feasible strategy for rapid generation of a high number of functional active T cells against Aspergillus from a single blood draw. Our data suggest that functionally active T cells against Aspergillus could be a promising treatment option for patients undergoing allogeneic SCT. (Blood. 2006;107: 2562-2569)

Description: Abstract 1234 Poster Board I-256 CLL is a heterogeneous disease with a variable clinical course. In this study the prognostic power of chromosome banding analysis (CBA), interphase FISH and IgVH status was evaluated. In total 399 untreated cases were analyzed. First, we could confirm the prognostic significance of established parameters such as age (≥65 yrs), white blood cell count (≥20.000/μl), IgVH status, TP53 deletion and 11q deletion in our cohort. In addition, a negative prognostic impact of translocations involving the IgH locus, especially t(14;18)(q32;q21) and of the complexity of the karyotype measured by the number of clonal chromosome aberrations in CBA was observed. Furthermore it became obvious that some parameters discriminated better for overall survival and other for time to treatment. While the impact of the IgVH status on overall survival was low within the first 5 years after diagnosis (mutated 88.5% surviving vs unmutated 82.0% surviving, log rank test p=0.022), an unmutated IgVH status was strongly correlated with a shorter median time to treatment (18.3 months unmutated vs 110.7 months mutated, log rank test p

Description: The chromosomal translocation t(12;21)(p13;q22) resulting in the TEL/AML1 (also known as ETV6/ RUNX1) fusion gene is the most frequent translocation in childhood B cell precursor (BCP) ALL. This type of ALL is characterized by a unique molecular signature, which includes the overexpression of the gene for the erythropoietin receptor (EpoR). So far, it is not known what causes the overexpression of the EpoR gene or whether it has any effect on the t(12;21) positive leukemia. We therefore aimed to evaluate potential mechanisms responsible for the upregulation of the EpoR in t(12;21) leukemias and to find out whether signalling via this receptor affects survival or proliferation of leukemic cells. In addition, we planned to explore signalling pathways linked to the respective effects and to elucidate relevant mechanisms that might be essential for cell survival. We first excluded the possibility that the EpoR expression is upregulated as a consequence of high Epo levels in the plasma that are induced by the patients’ low hemoglobin (Hb) levels. While Hb levels from patients with t(12;21)+ ALL were significantly lower compared to those with other subtypes of BCP ALL (median, 6,15g/dL and 7,9g/dL, respectively; p

Description: Abstract 750 Background: DKK1 is a negative regulator of the Wnt signaling pathway in bone that is overexpressed in a subset of newly-diagnosed MM patients with osteolytic lesions as well as in refractory and relapsed patients. Expression also correlates with the number of osteolytic lesions in untreated MM patients. BHQ880 is a novel anti-DKK1 human monoclonal antibody. Alleviation of DKK1 inhibition by BHQ880 results in activation of the Wnt signaling pathway, leading to increased bone mass mediated via upregulation of osteoblasts in mice and monkeys. In murine models of MM bone disease; this anabolic activity of BHQ880 increased trabecular and cortical bone density. Lytic bone disease in MM is caused by osteoclast activation and osteoblast inhibition. Current approved therapies for the treatment of MM bone disease are focused on osteoclast inhibition (e.g., bisphosphonates) and BHQ880 therapy may be able to reverse the effects of DKK1-induced osteoblast inhibition. Therefore, dual therapy with zoledronic acid to decrease bone resorption and BHQ880 to increase new bone formation may provide an effective treatment strategy for MM bone disease. Methods: In the phase I portion of this phase I/II study, patients with relapsed or refractory MM with prior skeletal-related event (SRE) were treated with BHQ880 as an IV infusion Q28 days. Patients also received Zol (4 mg) and approved MM Tx (bortezomib not allowed). Bone markers and total DKK1 levels along with bone mineral density are being measured. Full PK profiles were collected during the first and second cycle, after which predose (trough) samples were collected to assess accumulation. Results: Ten pts (6:M, 4:F), median age: 66.5 yrs (range 41- 70), performance status-0(5 pts), 1(4 pts), 2 (1 pts) have been enrolled in the following dose levels (mg/kg) 3 (2 pts), 10 (starting dose level -4 pts), 20 (4 pts) and have been on treatment for 1 day to 5 28-day cycles. No BHQ880-related AE's have been observed to date. Bone mineral density (BMD) data from the first two patients treated at 10 mg show the following: a.) Pt 1 (hip +5.8%, spine N/A due to surgery, wrist -1.8%); b.) Pt 2 (hip -0.2%, spine +6.1%, wrist not done). The biomarker data from these patients show: a.) Pt 1 shows a maximal +98% change over baseline in PINP at day 15 post-treatment, while osteocalcin (OC) changes +17% at day 15, reaching a +56% change over baseline at cycle 4 day 1; uNTx/Cr changes – 43 % at cycle 2 day 15. b) Partial data from Pt 2 suggests a -44 % change in PINP and –73 % change in OC and a +73% change in uNTx/Cr. Baseline total DKK1 levels on 3 patients ranged from 8.8 to 21.5 ng/mL. Preliminary PK analysis is available in 1 out of 3 patients treated at 10mg/kg. The Cmax achieved after the first infusion was 165 ug/mL and 201 ug/mL after the second infusion. Calculated t1/2 was 188 hours after the first infusion and 254 hours after the second infusion. Regarding overall exposure, AUC0-672 hours after first infusion was 36081 hr*ng/mL and 48533 hr*ng/mL after the second infusion. Conclusion: BHQ880 given IV Q28 days appears to be well tolerated in combination with Zol and chosen MM Tx. Once safety of 40 mg/kg BHQ880 or the MTD dose has been determined, pts will be enrolled in the phase II portion to assess activity on a SRE endpoint. Updated safety, efficacy, bone density and biomarker data will be presented on all patients at the upcoming ASH meeting. Disclosures: Padmanabhan: Genentech: Consultancy, Honoraria; GSK: Consultancy, Speakers Bureau; Celgene: Consultancy, Speakers Bureau. Off Label Use: BHQ880, a novel osteoblast activating, anti-DKK1 antibody. Dzik-Jurasz:Novartis: Employment, Equity Ownership. Gangolli:Novartis: Employment, Equity Ownership. Ettenberg:Novartis: Employment, Equity Ownership. Miner:Novartis: Employment, Equity Ownership. Bilic:Novartis: Employment, Equity Ownership. Whyte:Novartis: Employment, Equity Ownership. Mehdi:Novartis: Employment, Equity Ownership. Chiang:Novartis: Employment, Equity Ownership. Rae:Novartis: Employment, Equity Ownership. Shah:Novartis: Consultancy, Speakers Bureau; Celgene: Consultancy, Speakers Bureau; Millenium: Research Funding, Speakers Bureau; Amgen: Consultancy, Speakers Bureau; Elan: Consultancy. Giles:Novartis: Consultancy, Research Funding; BMS: Research Funding; Merck: Research Funding; Clavis: Research Funding. Stewart:Novartis: Consultancy; Amgen: Consultancy; Millenium: Consultancy, Research Funding; Proteolix: Consultancy; Celgene: Honoraria.

Description: There is no single sequence or breakpoint region linked to all patients with myeloma. It was thus elected to extract, amplify and fully sequence all (total) DNA present in circulating blood (CNA) using pyrosequencing with the Roche/454 sequencer (GSFLX). The origin of circulating DNA was investigated by local alignment analyses using BLAST and compared with the circulating DNA of 47 healthy individuals, aged 18 to 64 years. Thirty-one samples were subjected to total CNA sequencing during the course of the disease for a patient with lambda Bence Jones myeloma sequentially monitored from relapse, through complete response for 38 months and then with development of subsequent relapse. Conventional monitoring included both serum free light chain analyses and whole body CT-PET imaging. For comparative purposes the CNA was categorized by origin, functionality, chromosomal localization and genes, based on public databases (human reference genome build 36.2 and corresponding annotation file seq_gene.md as downloaded from NCBI (ftp.ncbi.nih.gov). The average length of sequenced nucleotides was 185.7 nucleotides and the total number of nucleotides sequenced per sample was 1.2 – 2.6 million. CNA sequences occurring with a frequency significantly greater or less than those observed for healthy controls were evaluated in detail. The next steps were to examine correlations with initial active myeloma, response to treatment, the complete remission state off all therapy, and subsequent relapse. The presence of 10 DNA sequences was highly correlated with the disease course: ZMYM2; TSPAN5; TLL1; PRKD1; ANTXR1; SYT14; PRKCH; MBNL1; EGFR and EDG7. Of these ZYMYM2 was highly correlated with disease reactivation and relapse off therapy. TSPAN5 showed a clear pattern with reduction to background levels during remission, but increase at the very earliest sign of relapse evident on CT-PET. Conversely, other sequences (GRID1; KIF16B) increased substantially during the peak impact of successful therapy. Multivariate analyses were used to identify the best combinations of gene sequences predictive of disease course and outcome. The combination of four genes (GRID1; PRKD1; ANTXR1; GAB1) was sufficient to define the early active disease data points vs the normal controls (odds-ratio OR: 135 [10.6 to 172]; p

Description: The clinical relevance of both minimal residual disease (MRD) and chimerism after hematopoietic stem cell transplantation (HSCT) for the detection of impending relapse has not yet been extensively studied. We investigated MRD in 119 consecutive children (median age, 12 years) with ALL (n=56), AML (n=36), MDS (n=20), or CML (n=7) who underwent bone marrow (n=80) or peripheral blood stem cell (n=39; T-cell depleted: n=18) transplantation in a single center. Thirteen patients received autologous HSCT and 106 patients underwent allogeneic HSCT. The donor was HLA-matched unrelated in 58 patients and HLA-identical related in 48 patients. The Wilms’ tumor gene (WT1) expression was used for the detection of MRD because WT1 gene is overexpressed in the vast majority of patients with leukemia. In contrast, WT1 gene is not expressed in normal peripheral blood mononuclear cells (PBMCs) and only weakly expressed in normal bone marrow (BM) cells. We performed a quantitative reverse transcriptase-polymerase chain reaction (PCR) to examine the level of WT1 gene expression. For the present study, we followed up MRD in both BM and PB after HSCT. In the 119 patients, a median of 11 analyses (range, 3–27 analyses) covering a median period of 708 days (range, 29–3101 days) was performed. Analysis of 25 paired BM and PB samples revealed that the level of MRD in BM was on average 9 times higher and paralleled that in PB. All 85 patients with continuous normal WT1 expression levels in BM and continuous undetectable WT1 expression levels in PB remained in complete remission without relapse at a median of 1884 days (range, 58–5905 days) after HSCT. In contrast, all 32 patients who suffered from hematological relapse after a median of 152 days (range, 28–1104 days) presented with high levels of WT1 gene expression in BM and PB (P 〈 .001). In 19 patients, we observed a gradual or rapid increase of WT1 expression levels at a median of 31 days (range, 12–119 days) before hematological relapse. In 14 of these 19 patients, DNA was available at the time of increase of WT1 expression level to perform analysis of hematopoietic chimerism using a semi-quantitative short-tandem-repeat PCR. Interestingly, 10 of the 14 patients (71%) revealed a complete donor chimerism, whereas only 4 of the 14 patients (29%) showed a mixed chimerism. In two patients, we diagnosed a molecular relapse using WT1 gene expression at 161 and 360 days after transplantation. At the time of molecular relapse, both children revealed a complete donor chimerism. In both patients, molecular remission was achieved by withdrawal of cyclosporin A and by donor lymphocyte transfusion, respectively. Both children are alive and well without relapse at 10 and 9 years after transplantation. In conclusion, quantitative analysis of WT1 gene expression is a valuable tool for monitoring of MRD in patients with ALL, AML, CML, and MDS after HSCT. Increasing levels of WT1 gene expression strongly predict hematological relapse. Therefore, this approach is very useful for early diagnosis and treatment of molecular relapse after HSCT. MRD measurement using WT1 gene expression is more sensitive for the detection of impending relapse than the analysis of hematopoietic chimerism.