ALBERT

Description: Purpose: The GHSG HD9 trial had established BEACOPP escalated (BE) as new standard of care for advanced-stage HL patients by showing significant superiority in terms of failure-free survival (FFTF) and overall survival (OS) over COPP/ABVD and BEACOPP baseline (BB) (each 8 cycles). The successor study, HD12, evaluated a possible reduction in toxicity by comparing 8 cycles of BE with 4 cycles BE followed by 4 cycles BB. The second question in this trial related to the need of additional radiotherapy (RT) to initial bulk and residual disease. Patients and methods: HL patients in stage IIB with large mediastinal mass and/or E-lesions or stage III/IV were randomised according to a 2×2-factorial design between: 8BE + RT, 8BE no RT, 4BE+4BB + RT, 4BE+4BB no RT. Reviewing CT-images before and after chemotherapy treatment, fields for RT were centrally planned by a multidisciplinary diagnostic panel blinded for the randomisation arm. Primary endpoint of the trial was FFTF. Between 9/1999 and 1/2003, a total of 1,670 patients aged 16–65 were randomized. For this final analysis at a median follow up of 78 months, 99 patients were excluded (42 HL not confirmed, 20 revision of stage, 20 no study treatment or documentation, 17 others) resulting in 1,571 eligible patients. Results: Patient characteristics in the 4 groups were comparable with 49% of patients in stage III, 35% in stage IV, 68% reporting B-symptoms and 28% having a large mediastinal tumor. An IPS of 3 or greater was reported for 38% of patients, predominant histology was nodular sclerosis with 57% of cases. Treatment-related toxicity of WHO grade III/IV was observed in 97% of patients. Most prominent differences between pooled chemotherapy arms were anemia (65% 8BE vs 51% 4BE+4BB) and thrombopenia (65% vs 51%). Treatment outcome: complete remission 92.4%; early progression 2.2%; progression/relapse 7.8% (6.6% and 8.5%). A total of 156 (9.9%) deaths (72 vs 84) have been observed (22 vs 32 acute or salvage treatment toxicity; 15 vs 24 HL; 22 vs 13 secondary neoplasia). Most treatment related deaths occurred in the 〉60 years age group, the first 4 cycles and the IPS〉 3 RF groups. Secondary neoplasias were observed in 77 patients (4.9%): AML/MDS 1.5% vs 1.4%, NHL 1.4% vs 0.6% and solid tumors/others 2.5% vs 2.3%. At 5 years, OS was 91%, FFTF 85.5% and progression free survival (PFS) 86.2% (Kaplan- Meier estimates). Estimates for the difference at 5 years are 1.8% for OS, 2.3% for FFTF and 2.7% for PFS favoring BE. However, there was no statistical difference between 8x BE and 4BE+4BB in all outcome parameters (p〉0.19, log rank test). There is also no significant difference between the RT or no-RT arms in this study with the caveat that a number of high-risk patients receiving RT based on the blinded panel decision. Conclusion: The adoption of 4BE+4BB as a new standard in the future GHSG studies will depend on a refined analysis of the total data set and will be presented.

Description: Coagulation activation by tissue factor (TF) is implicated in cancer progression, cancer-associated thrombosis and metastasis. The role of direct TF signaling pathways in cancer, however, remains incompletely understood. Here we address how TF contributes to primary tumor growth by using a unique pair of isotype-matched antibodies that inhibit either coagulation (monoclonal antibody [Mab]-5G9) or direct signaling (Mab-10H10). We demonstrate that the inhibitory antibody of direct TF-VIIa signaling not only blocks TF-VIIa mediated activation of PAR2, but also disrupts the interaction of TF with integrins. In epithelial and TF-expressing endothelial cells, association of TF with β1 integrins is regulated by TF extracellular ligand binding and independent of PAR2 signaling or proteolytic activity of VIIa. In contrast, α3β1 integrin association of TF is constitutive in breast cancer cells and blocked by Mab-10H10 but not by Mab-5G9. Mab-5G9 has antitumor activity in vivo, but we show here that Mab-10H10 is at least as effective in suppressing human xenograft tumors in 2 different models. Breast tumor growth was also attenuated by blocking PAR2 signaling. These results show that tumor cell TF-PAR2 signaling is crucial for tumor growth and suggest that anti-TF strategies can be applied in cancer therapy with minor impairment of TF-dependent hemostatic pathways.

Description: Activating mutations in Flt3, N- and K-Ras have been reported in all AML subtypes and represent common molecular defects in de novo AML. We have previously shown that these mutations lead to constitutive AKT phosphorylation and activation. As a consequence, Akt phosphorylation is found in myeloid blasts of the majority of AML patients. We reasoned that constitutively active AKT may contribute to leukemia development, and therefore we assessed the contribution of AKT in oncogenic transformation in vivo. For this purpose, we established an inducible mouse model expressing myristylated AKT1 under the control of the scl-3′ enhancer (MyrAKT1). This system restricts activated AKT1 to endothelium, hematopoietic stem cells and myeloid lineage cells at a low but detectable level. About 40% of induced mice developed a myeloproliferative disorder after latencies of 7 to 22 months. Onset of disease was frequently associated with hemangioma formation, due to endothelial MyrAKT1 expression. The myeloproliferative disorder was associated with splenomegaly with increased extramedullary hematopoiesis, while the peripheral blood contained mature granulocytes. Furthermore, the stem cell and progenitor cell compartment in spleens and bone marrow of these mice was altered compared to control mice. Colony formation assays with MyrAKT1-expressing bone marrow suggested that overactivation of AKT1 enhanced proliferation. The AKT1-induced disease was transplantable by both bone marrow and spleen cells. These findings highlight the oncogenic capacity of constitutively activated AKT1 in vivo and indicate that AKT is an attractive target for therapeutic intervention in AML.

Description: The α-globin Constant Spring (CS) mutation (α142 STOP → Gln; TAA → CAA) is the most prevalent non-deletional thalassemia in south East Asia and southern China. DNA diagnosis of Hb H Constant Spring (Hb H CS; 2 α-gene deletions and 1 CS mutation) is often required because it can be missed by electrophoresis. The clinical phenotype of Hb H CS is more severe than classical Hb H disease. We sought to characterize the unique hematological and clinical features of Hb H CS patients, especially compared to those with Hb H disease, who were identified through the Thalassemia Clinical Research Network (TCRN). A total of 836 patients enrolled in the TCRN registry in Canada and the U.S. (2001 to 2005) were screened for this analysis. Genotyping of 836 thalassemia patients identified 106/836 (12.7%) with Hb H and 46/836 (5.5%) with Hb H CS. 2 had other non-deletional mutations. Among Hb H CS patients, 48% were female; mean age was 13±11 years. Among patients who had spleens, splenomegaly was more prevalent in Hb H CS than Hb H patients (16% vs 1%, p=0.001). Among Hb H CS patients who had splenomegaly, average spleen span was 3.67 ± 2.25cm. 13% of the Hb H CS and 2% of the Hb H patients had their spleen removed (p=0.005). Mean Hb level was higher in the splenectomized Hb H CS patients than in the non-splenectomized Hb H CS patients (9.85 ± 2.25 g/dL vs. 8.25 ± 0.76 g/dL, p=0.006). Post-splenectomy portal vein thrombosis was reported in 1 Hb H CS patient. 7.5% (3/40) of non-splenectomized Hb H CS patients had bacteremia or infections requiring intravenous antibiotics. 8.7% (4/46) of Hb H CS patients underwent cholecystectomy. 24% of HbH CS and none of the Hb H patients were placed on regular transfusions (〉8/year) and chelation therapy. Mean age of initiation of transfusions was 3.5±1.3 years (range 2–5 years). Mean ferritin level was higher in the non-transfused Hb H CS patients than in the Hb H patients (369.9 ± 409.9 ng/ml vs 175.9 ± 304.2 ng/ml, p=0.01), suggesting increased gastrointestinal iron absorption in Hb H CS. In 5 transfused Hb H CS patients, liver iron concentration was obtained showing elevated levels (27.2 ± 13.9 μgm/gm dry wt). Growth delay was more apparent in the Hb H CS patients (n=19) compared to 20 Hb H patients (Mean height Z score: −1.34 ± 0.98 for Hb H CS vs −0.82 ± 1.15 for Hb H (p=0.16) and mean weight Z score: −1.15 ± 0.88 for Hb H CS vs. −0.83 ± 1.61 for Hb H (p=0.47)). Bone density scans in Hb H CS patients revealed a higher prevalence of low bone mass than that detected in Hb H patients: mean L1-L4 spine Z/T-score of −1.60 ± 0.86 vs. −0.93 ± 0.80 (p=0.02). 40% (4/10) of adult female Hb H CS patients had 1 or more successful pregnancies, some requiring transfusion support through pregnancy. Patients with Hb H CS have a severe phenotype of α-thalassemia. They have moderately severe anemia, which sometimes requires regular transfusions, splenectomy, or both. Patients with Hb H CS commonly have iron overload, growth delay, and reduced bone mass. Early diagnosis in the neonatal period, regular monitoring, and appropriate treatment considerations for initiation of regular transfusions are key.

Description: Purpose: Hodgkin Lymphoma (HL) is one of the best curable cancers in adults. Thus, current research is focusing on the increasing proportion of HL survivors with respect to the long-term consequences of therapy. Especially health related quality of life (QoL) needs thorough investigation since this is relevant for HL patients, complex and so far there is limited knowledge. QoL incorporates different aspects such as general QoL, fatigue, emotional, physical, role, social, sexual and cognitive functions. This analysis is focusing on QoL in the HD10–12 trials of the GHSG with special emphasis on cognitive functions (CF) and possible predictors in a 2 year follow-up. Methods: Patients of the GHSG trials HD10–12 completed the QLQ-C30, the MFI20 and some additional items at the time of diagnosis, after chemotherapy, after radiotherapy and at follow-up examinations. We describe the courses of the QLQ-C30 scales with means and 95%-confidence intervals for each measurement point and in relation to the respective norm values. In accordance with well established criteria, we used a cut off point of 10 points below the expected value to define self reported cognitive deficits (CD). In multiple regressions we analysed the role of the following predictors for cognitive functions 2 years after treatment: age, sex, disease stage, treatment modality, baseline score of cognitive functioning and symptoms of peripheral neuropathy (PNP) before and after chemotherapy application. Results: In the sample of 3608 patients all scales showed abnormal values from the beginning, further deterioration to the end of chemotherapy and continuous recovery from the end of radiotherapy on. Most scales improved clearly beyond their pre-treatment values but only physical functioning finally approached normal values. The CF scale showed a remarkable flat long term course and, opposed to the other scales, reached no improvement beyond the pre-treatment level. In 1044 patients with baseline and 2 year results, 439 met the criteria of relevant CD at baseline. Of these, 60.4% still felt handicapped after 2 years and 39.6% were in the range of normal values. 605 patients reported no CD at baseline and 69.6 % of them remained unaffected after 2 years (30.4% had developed CD). After adjustment for age, sex, disease stage, treatment modality and CF baseline scores, the regression analyses showed significant impact of symptoms of PNP before (p

Description: The Epstein-Barr virus (EBV) protein LMP1 is considered to be a functional homologue of the CD40 receptor. However, in contrast to the latter, LMP1 is a constitutively active signaling molecule. To compare B cell–specific LMP1 and CD40 signaling in an unambiguous manner, we generated transgenic mice conditionally expressing a CD40/LMP1 fusion protein, which retained the LMP1 cytoplasmic tail but has lost the constitutive activity of LMP1 and needs to be activated by the CD40 ligand. We show that LMP1 signaling can completely substitute CD40 signaling in B cells, leading to normal B-cell development, activation, and immune responses including class-switch recombination, germinal center formation, and somatic hypermutation. In addition, the LMP1-signaling domain has a unique property in that it can induce class-switch recombination to IgG1 independent of cytokines. Thus, our data indicate that LMP1 has evolved to imitate T-helper cell function allowing activation, proliferation, and differentiation of EBV-infected B cells independent of T cells.

Description: Although the phosphatidylinositide 3-kinase (PI3K)/Akt pathway has been reported to contribute to the malignant growth of multiple myeloma (MM), the true relevance of Akt kinases for this disease is still unclear. In particular, functional analyses in primary tumor cells and genetic target validation experiments are missing. Here, we used combined functional and molecular analyses to determine the importance of Akt activity in a large panel of primary MM samples and in MM cell lines. Akt down-regulation with isoform-specific siRNA constructs or with an Akt1/2-specific pharmacologic inhibitor strongly induced apoptosis in approximately half of the primary MM samples analyzed. Sensitivity to Akt inhibition strongly correlated with the activation status of Akt as determined by immunohistochemistry, phospho-Akt–specific flow cytometry, and Western analysis. Additional blockade of the MAPK and the IL-6R/STAT3 pathways was often not sufficient to decrease the viability of MM cells resilient to Akt inhibition. Taken together, these experiments led to the identification of 2 myeloma subgroups: Akt-dependent and Akt-independent MM.

Description: The translocation t(15;17) generates the chimeric PML-RARα transcription factor that is the initiating event of acute promyelocytic leukemia. A global view of PML-RARα transcriptional functions was obtained by genome-wide binding and chromatin modification analyses combined with genome-wide expression data. Chromatin immunoprecipitation (ChIP)–chip experiments identified 372 direct genomic PML-RARα targets. A subset of these was confirmed in primary acute promyelocytic leukemia. Direct PML-RARα targets include regulators of global transcriptional programs as well as critical regulatory genes for basic cellular functions such as cell-cycle control and apoptosis. PML-RARα binding universally led to HDAC1 recruitment, loss of histone H3 acetylation, increased tri-methylation of histone H3 lysine 9, and unexpectedly increased trimethylation of histone H3 lysine 4. The binding of PML-RARα to target promoters and the resulting histone modifications resulted in mRNA repression of functionally relevant genes. Taken together, our results reveal that the transcription factor PML-RARα regulates key cancer-related genes and pathways by inducing a repressed chromatin formation on its direct genomic target genes.

Description: Herbs have successfully been used in traditional Chinese medicine for centuries. However, their curative mechanisms remain largely unknown. In this study, we show that Wogonin, derived from the traditional Chinese medicine Huang-Qin (Scutellaria baicalensis Georgi), induces apoptosis in malignant T cells in vitro and suppresses growth of human T-cell leukemia xenografts in vivo. Importantly, Wogonin shows almost no toxicity on T lymphocytes from healthy donors. Wogonin induces prolonged activation of PLCγ1 via H2O2 signaling in malignant T cells, which leads to sustained elevation of cytosolic Ca2+ in malignant but not normal T cells. Subsequently, a Ca2+ overload leads to disruption of the mitochondrial membrane. The selective effect of Wogonin is due to its differential regulation of the redox status of malignant versus normal T cells. In addition, we show that the L-type voltage-dependent Ca2+ channels are involved in the intracellular Ca2+ mobilization in T cells. Furthermore, we show that malignant T cells possess elevated amounts of voltage-dependent Ca2+ channels compared with normal T cells, which further enhance the cytotoxicity of Wogonin for malignant T cells. Taken together, our data show a therapeutic potential of Wogonin for the treatment of hematologic malignancies.

Description: Background: Approximately 20% of patients diagnosed with Hodgkin lymphoma (HL) are more than 60 years of age. These elderly patients still have a poor prognosis, especially when presenting with advanced stages and higher age. The main reason is underdosing of treatment, which is due to reduced tolerability of chemotherapy and age-related comorbidities. In the GHSG experience, elderly patients in the HD9 trial did not profit from the BEACOPP regimen in terms of overall survival, though a better HL specific freedom from treatment failure was achieved as compared to COPP/ABVD. Thus, the GHSG has developed the BACOPP regimen (bleomycin, doxorubicin, cyclophosphamide, vincristine, procarbazine, and prednisone), in which etoposide was omitted to improve tolerability. Further modifications were a 1 week pretreatment phase with vincristine and prednisone, a limitation of vincristine in patients older than 65 years, an increase of the anthracyclines dose, and concomitant application of erythropoietin. Here we report on the final analysis of this multi-center phase II study for elderly patients. Methods: Between 2004 and 2005, 65 patients with HL in intermediate or advanced stages aged between 60 and 75 years were recruited. Treatment consisted of 6 cycles BACOPP in patients achieving a complete remission (CR) after 4 cycles or 8 cycles BACOPP in case of PR (partial remission) after 4 cycles. The primary endpoints were protocol adherence and response rates. Secondary endpoints included WHO grade III/IV toxicities, Kaplan Meier estimates of progression free survival (PFS), freedom from treatment failure (FFTF), and overall survival (OS). Results: Sixty patients (92%) were eligible for the final analysis. The majority of treatment courses (75%) were administered according to protocol. However, there was a tendency towards reduced dosing in cycles 5 to 8, especially for patients who had reached a CR after 4 cycles of BACOPP. In total, 51 patients showed CR/CRu (85%), 2 PR (3%) and 4 progression of disease (7%). Survival estimates and their 95% confidence intervals are shown in table 1. Table 1. Kaplan-Meier rates and 95% confidence intervals (CI) for FFTF, PFS and OS. time point rate (%) CI (95%) FFTF 12 months 73 61–84 24 months 67 55–79 PFS 12 months 75 64–86 24 months 68 56–80 OS 12 months 85 76–94 24 months 76 65–87 WHO grade III–IV toxicities were documented in 52 patients (87%). With a median observation time of 33 months, 18 deaths (30%) have been observed. Seven therapy associated fatal outcomes were documented. Conclusion: The new BACOPP regimen developed for elderly HL patients shows a high CR rate (85%). The FFTF rate at 2 years is within the range known from other schedules in this patient cohort. Overall, the regimen is feasible, but the therapy-associated death rate was high in our patient cohort. Thus, further studies and new approaches are still needed to substantially improve the outcome of elderly patients with early unfavorable or advanced stage HL.