ALBERT

Description: To study outcomes of adults with idiopathic thrombocytopenic purpura (ITP), we performed a follow-up study in a cohort of 152 consecutive patients who were treated according to a well-defined algorithm. Long-term outcomes were determined relative to the response 2 years after diagnosis, because most (93%) patients who ultimately attained platelet counts above 30.0 × 109/L (30 000/μL) did so within this time frame. Complete follow-up for mortality could be studied in 99% of patients and for morbidity in 95% of patients, with a mean of 10.5 years. Within 2 years after diagnosis, 4 patients died, 2 were lost to follow-up, and 12 were reclassified as having secondary immune thrombocytopenia. Of the remaining 134 patients, 114 (85%) had obtained platelet counts above 30.0 × 109/L while all therapies had been discontinued. These patients had a long-term mortality risk equal to the general population. Twelve of 134 patients (9%), all with severe thrombocytopenia, had refractory disease and suffered a mortality risk of 4.2 (95% confidence interval, 1.7-10.0). Bleeding and infection equally contributed to the death of these patients. Another 8 patients (6%) had platelet counts above 30.0 × 109/L while on maintenance therapy. Similar to patients with refractory disease, these latter patients had considerably increased ITP-related hospital admissions, but mortality was only slightly higher than in the general population. In conclusion, most adults with ITP have a good outcome with infrequent hospital admissions and no excess mortality. The absence of gross morbidity and mortality in patients with moderate thrombocytopenia supports clinical practice refraining from further treatment.

Description: A functional BCR is essential for survival of normal B-cells, in response to cognate antigen or tonic, antigen-independent stimuli. Cross-linking of BCR by antigen triggers key phosphorylation events in the early signalosome. Mammalian wild type BRAF, a member of the RAF cytosolic kinase family (A-RAF, B-RAF, C-RAF) is critical to BCR function, mediating RAS-RAF-MEK-ERK or mitogen-activated protein kinase (MAPK) pathway signal transduction, via phosphorylation of ERK1/2. The MAPK pathway is a central conduit for survival and proliferation. Importantly, use of inducible deletion models have shown that wild type BRAF is the predominant kinase that transduces BCR signals to activate ERK1/2, with C-RAF playing an accessory role and A-RAF displaying a x20-30 fold lower basal activity in ERK1/2 stimulation. Tonic BCR survival signals in normal B-cells are also transduced via phosphorylation of ERK1/2, but do not require antigen stimuli. Whether the BCR plays a comparable role in sustaining survival in malignant B-cells is less well characterized and remains a central question in understanding the origins and progression of mature B-cell neoplasms. Hairy cell leukaemia (HCL) is a rare B cell leukaemia with characteristic hair-like cytoplasmic projections on tumor cells, displaying distinctive activation markers. A striking feature of monoclonal HCL tumors is expression of multiple variant immunoglobulin heavy chain (sIgH) isotypes as components of the BCR on individual tumor cells, defining the major subset of disease (mult-HCL). Most mult-HCL tumors exhibit IGV somatic hypermutation with a low level of on-going mutations and AID expressed, implicating initial contact with antigen via the BCR. The functional relevance of the BCR to transformation and survival however has as yet not been mapped in HCL. Seminal exome sequencing data in typical HCL identified mutant BRAF V(600)E as almost universal in this tumor. Consistent with mutant BRAFV(600)E, levels of phosphorylated ERK (p-ERK) are raised in hairy cells. As there is an essential requirement for wild type BRAF in transducing BCR signals, the question arises how mutant BRAF may affect BCR function in HCL, given requirements for dimerization of wild type BRAF for ERK1/2 activation, and whether ERK1/2 activation can be enhanced by functional BCR signals for downstream effector signals in HCL. To examine this, we evaluated BCR signalling in mult-HCL (n=10), in cases uniformly displaying mutated BRAF and IGHV genes. Two apparent functional sets were delineated by IgD co-expression. In sIgD+ mult-HCL, IgD mediated persistent Ca2+ flux, which was also evident via 〉1 sIgH isotype, linked to increased ERK1/2 activation and BCR endocytosis (4/4 cases). In sIgD-vemult-HCL however (6/6 cases), BCR-mediated signals activating ERK1/2 for downstream effects were restricted to a single sIgH isotype, with sIgM notably dysfunctional and remaining immobilised on the cell surface. These observations reveal a clear discordance between expression and function of individual isotypes in mult-HCL. In dual sIgL expressing mult-HCL (3/3 cases), only a single sIgL was fully functional. We next examined effects of anti-BCR stimuli on mult-HCL tumor cell survival ex-vivo. Significantly, all functional non-IgD isotypes increased ERK1/2 phosphorylation but triggered apoptosis of tumor cells, in both sets (4/4 cases) to establish a consistent outcome in these replicate cases. IgD stimuli, in marked contrast retained tumor viability with no evidence for pre-apoptotic effects (2 cases; 1 mult-HCL and 1 sIgD only-HCL). Despite mutant BRAF, BCR signals amplify ERK1/2 phosphorylation, but isotype dictates functional downstream outcomes. In mult-HCL lacking IgD, a role for antigen in stimulating the BCR for tumor persistence appears unlikely. It remains feasible that mutant BRAF in these cases retains tonic signals through activated ERK1/2 for survival, but this remains speculative at present. Surface IgD emerges with potential to transduce BCR signals for tumor survival and persistence in-vivo, but given its enigmatic role even in normal B-cells, its relevance to HCL progression is unclear. These observations raise the possibility that mutant BRAF may be a mechanism to bypass any antigen-dependent BCR signalling constraints in mult-HCL. Disclosures: No relevant conflicts of interest to declare.

Description: Abstract 880 On behalf of the German Low-Grade Lymphoma Study Group (GLSG) and the European MCL Network Background: In younger patients with mantle cell lymphoma (MCL), autologous stem cell transplantation (ASCT) significantly prolonged response duration with a trend towards improved overall survival in a randomized trial of the European MCL Network (Dreyling et al., ASH 2008). A recently updated clinical trial of the German Low-Grade Lymphoma Study Group (GLSG) also reported a significantly prolonged response duration by the addition of Rituximab to first-line CHOP (Hoster et al., ASH 2008). By pooled analysis of these trials we investigated whether Rituximab and ASCT independently prolong response duration. Methods: The analysis included all advanced stage MCL patients of the trials “CHOP vs. MCP” (Nickenig et al., Cancer 2006), “CHOP vs. R-CHOP” (Lenz et al., JCO 2005) and European MCL trial 1 (Dreyling et al., Blood 2005) with complete or partial remission to either CHOP or R-CHOP first-line induction and randomization between ASCT and Interferon-α maintenance. Response duration was defined as time from the end of successful induction chemotherapy to relapse or death from any cause, overall survival from the end of successful induction chemotherapy to death from any cause. Stratified Kaplan-Meier curves for response duration and overall survival were calculated for the four treatment groups (CHOP without ASCT, CHOP with ASCT, R-CHOP without ASCT and R-CHOP with ASCT). By multiple Cox regression we tested whether the impact of Rituximab (R) and ASCT was independent and additive. Results: One-hundred and eighty patients with MCL were evaluable, 80 treated with R-CHOP, 78 with ASCT (CHOP without ASCT: 56, CHOP with ASCT: 46, R-CHOP without ASCT: 44 and R-CHOP with ASCT: 34). Median age was 55 years (range 34-65), the MIPI classified 71% as low risk, 22% as intermediate risk, and 6% as high risk, and baseline characteristics were comparable between treatment groups. With a median follow-up of 63 months, median response duration was 16 months after CHOP without ASCT, 26 months after R-CHOP without ASCT, 39 months after CHOP with ASCT, and 41 months after R-CHOP with ASCT. In multiple Cox regression including R and ASCT, the hazard ratios of R (0.60, 95% CI 0.42-0.86, p = 0.0056) and ASCT (0.50, 95% CI 0.35-0.70, p = 0.0001) were independently significant. There was no interaction between R and ASCT (p=0.43). Median overall survival was 54 months after CHOP without ASCT, 66 months after R-CHOP without ASCT, 90 months after CHOP with ASCT, and not reached after R-CHOP with ASCT. The hazard ratios for OS were 0.70 (95% CI 0.44-1.12, p = 0.14) for R and 0.63 (95% CI 0.41-0.97, p = 0.0379) for ASCT. Conclusions: Our results indicate an additive effect of ASCT and Rituximab in combination with CHOP on response duration in advanced stage MCL patients and support strategies of several study groups to combine Rituximab-containing induction therapies with ASCT in younger MCL patients. However, even after combined treatment approaches, delayed relapses have been observed, supporting the use of maintenance therapy and the introduction of molecular targeted therapeutical strategies. Disclosures: Hoster: Roche: Travel Support. Off Label Use: Rituximab in Mantle Cell Lymphoma. Pfreundschuh:Roche: Membership on an entity's Board of Directors or advisory committees. Dührsen:Roche: Honoraria, Research Funding; Amgen: Honoraria, Research Funding. Gisselbrecht:Roche: Research Funding, Speakers Bureau. Unterhalt:Roche: Travel Support. Dreyling:Roche: Honoraria, Research Funding.

Description: Patients with the B-cell malignancy hairy cell leukemia (HCL) exhibit a skewed T-cell repertoire with oligoclonal expression or absence of many members of the T-cell receptor (TCR) BV gene families. To evaluate whether interferon-α (IFN-α) therapy would not only restore normal hematopoiesis, but also the abnormal T-cell repertoire, we studied T lymphocytes from a cohort of HCL patients treated by IFN-α in the past, at initiation, and at several intervals up to 6 years of IFN-α treatment. The junctional regions from 22 TCRBV gene families were analyzed after polymerase chain reaction amplification of cDNA (RT-PCR) using family specific primers. In all seven patients improvement of the skewed T-cell repertoire was not seen until 2 years of treatment. It consisted of disappearance of oligoclonal subpopulations and (polyclonal) reappearance of absent TCRBV gene families. The RT-PCR results were correlated with the TCRBV protein expression using TCRBV-specific monoclonal antibodies. T lymphocytes from four patients with active HCL contained large expansions of particular TCRBV-expressing cells (up to 25% of the CD3+cells; 600 to 700/μL whole blood), which decreased during IFN-α therapy in both patients tested. Finally, restoration of the TCR repertoire matched normalization of the functional immune repertoire as measured by proliferative, helper, and cytotoxic T-lymphocyte precursor frequencies against major histocompatibility complex–unrelated individuals. In conclusion, oligoclonal bands of TCRBV gene families found by RT-PCR correspond with a dramatic increase in circulating T lymphocytes expressing the same TCRBV family. Moreover, IFN-α can restore the skewed T-cell repertoire and suppress persistent T-cell clones upon treatment of the accompanying malignancy.

Description: Immunoglobulin class switching usually involves deletion of part of the immunoglobulin CH region. By DNA fiber fluorescence in situ hybridization (FISH) with a barcode of probes covering the DH, JH, and CH genes, the configuration of the entire CH region can be visualized on single DNA molecules. Using this technique, we have studied class switching in three types of B-cell neoplasia, mantle-cell lymphoma (MCL), follicular lymphoma (FL) and hairy cell leukemia (HCL), representing B cells in, respectively, pregerminal center, germinal center, and postgerminal center stages of development. In MCL and FL, simultaneous detection of the t(11;14) and t(14;18) breakpoint with probes for the BCL-1 and BCL-2 loci, respectively, allowed differentiation between productive and nonproductive alleles. In none of 10 MCL cases was class switching detected. In 21 HCL, all nonimmunoglobulin M (IgM) cases had class-switch deletion consistent with the expressed isotype on at least one allele. In FL, however, a peculiar pattern of CH rearrangement was observed. In IgM expressing FL, the translocated alleles had switched in 11 of 13 cases, and the nontranslocated allele showed complex rearrangements downstream from the Cμ-Cδ genes in 9 of 13 cases. These downstream rearrangements may reflect tumor-specific deregulation of the class-switch machinery. All seven immunoglobulin G (IgG) expressing FL showed class switching on both alleles. Fiber FISH analysis also showed several polymorphisms. The most frequent one, present on 38% of all analyzed alleles, consisted of an extra Cγ gene or pseudogene in the 3′ cluster. © 1998 by The American Society of Hematology.

Description: A family cohort study was performed to assess the absolute risk of thromboembolism associated with inherited protein S (PS) deficiency type I and type III. Probands were consecutive patients with thromboembolism and either type I or type III deficiency. Of living first degree relatives (age〉15 years), 156 (90%) from type I deficient probands (cohort 1) and 268 (88%) from type III deficient probands (cohort 2) were analyzed. Annual incidences of venous thromboembolism were 1.47 and 0.17 in deficient and non-deficient relatives in cohort 1 (RR 8.9; 95% CI 2.6–30.0), and 0.27 versus 0.24 in cohort 2 (RR 0.9; 95% CI 0.4–2.2). The cut off level of free PS to identify subjects at risk was 30%. Lower levels were found in 40% and 1% of relatives in cohort 1 and 2, respectively. We demonstrated that the cut-off level of free PS is considerably lower than the lower limit of its normal range in healthy volunteers, that is commonly used (in our study 30% in stead of 65%). If we had used only a total PS assay, 8 (2%) relatives at risk would have been missed, but 67% (346/517) of relatives would not be tested. Only testing for free PS deficiency (free PS levels

Description: We used biopsy specimens of primary nodal diffuse large B-cell lymphoma (DLBCL) to investigate whether the inhibition of caspase 8 and/or 9 apoptosis signaling pathways predicts clinical outcome. Expression levels of cellular FLICE inhibitory protein (c-Flip) and numbers of active caspase 3-positive lymphoma cells were used to determine the status of the caspase 8-mediated pathway. Expression levels of Bcl-2 and X-linked inhibitor of apoptosis (XIAP) were used to determine the status of the caspase 9-mediated pathway. Expression of c-Flip, XIAP, Bcl-2, and caspase 3 activity all provided prognostic information. According to these immunohistochemical parameters, inhibition of either or both caspase signaling pathways was detected in all patients. Three groups of patients were identified, one with a caspase 8 inhibition profile, one with caspase 8 and 9 inhibition profiles, and one with a caspase 9 inhibition profile. Caspase 9 inhibition was strongly associated with poor response to chemotherapy and usually with fatal outcome, whereas caspase 8 inhibition was associated with excellent clinical outcome. Thus, our data strongly suggest that inhibition of the caspase 9-mediated pathway, but not the caspase 8-mediated pathway, is a major cause for therapy resistance in patients with nodal DLBCL.

Description: Achieving a complete cytogenetic response (CCgR) is a major target in the treatment of chronic myeloid leukemia (CML) with interferon-α (IFN-α), but CCgRs are rare. The mean CCgR rate is 13%, in a range of 5% to 33%. A collaborative study of 9 European Union countries has led to the collection of data on 317 patients who were first seen between 1983 and 1997 and achieved CCgRs with IFN-α alone or in combination with hydroxyurea. The median time to first CCgR was 19 months (95% CI, 17-21; range, 3-84 months). At last contact, 212 patients were still alive and in continuous CCgR; 105 patients had lost CCgR, but 53% of them were still alive and in chronic phase. IFN-α treatment was discontinued permanently in 23 cases for response loss, in 36 cases for chronic toxicity (15 are still in unmaintained continuous CCgR), and in 8 cases because it was believed that treatment was no longer necessary (7 of these 8 patients are still in unmaintained continuous CCgR). The 10-year survival rate from first CCgR is 72% (95% CI, 62%-82%) and is related to the risk profile. High-risk patients lost CCgR more frequently and more rapidly and none survived more than 10 years. Low-risk patients survived much longer (10-year survival probability 89% for Sokal low risk and 81% for Euro low risk). These data point out that a substantial long-term survival in CCgRs is restricted mainly to low-risk and possibly intermediate-risk patients and occurs significantly less often in high-risk patients.

Description: Imatinib mesylate (IM) induces a high rate of cytogenetic remissions in first chronic phase CML, but molecular remissions are rare. A recent phase II study suggested a higher rate of molecular remissions upon treatment with 800 mg IM (Cortes et al. Blood 2004). Cytarabin (Ara-C) is an active drug in myeloid leukemias, especially when intensified dosages are applied. In-vitro studies of IM and Ara-C have been shown synergistic anti-proliferative effects. With the aim to prevent resistance and to induce an early, high rate of molecular remissions, we set out to evaluate the combination of IM and Ara-C in a dose-escalation study of consecutive cohorts treated with daily IM at dosages of 200 mg, 400 mg, 600 mg or 800 mg combined with intravenous Ara-C, added as 2 consecutive cycles of i.v. therapy at days 1–7 at a dose of 200 mg/m2/24 hrs or 1,000 mg/m2/24 hrs. Primary endpoints are dose-limiting-toxicity (DLT) and quantitative molecular response as assessed by standardized real-time Taqman PCR of bcr/abl transcripts. Patients without a major molecular response after 12 months are evaluated for mutations by direct sequencing. From the start in August 2001, thusfar 127 pts are included in cohorts I through V. The median age is 48 (20–66) years. Sofar, 2 DLTs were observed following evaluation of 112 pts, including reversible grade IV CZS toxicity (cohort IIIb, Ara-C 1,000/IM 400) and one pneumonia, complicated by lethal respiratory failure in cohort IIIa (Ara-C 200/IM 600). The median number of neutropenic (4.5 log) molecular response were, respectively, 51% (±7) and 28% (±7) at 18 months from the start of treatment. Probabilities for major and complete cytogenetic response rates were 83% and 67%, respectively, at 18 months. Overall survival was 99 (±1) at 18 months and progression-free survival was 94% (±3). Two patients developed accelerated disease, 1 patients lost a complete hematological response. Among 30 pts without a major molecular response at 12 months, 2 pts acquired a point mutation of the Abl kinase domain resulting in amino-acid substitutions Phe359→ Val and Glu459→ Lys, respectively. These results suggest that the combination of escalated Imatinib combined with standard or intensified Ara-C may prevent resistance and mirrors the synergy of both drugs that was found in-vitro.

Description: Abstract 2642 Introduction With the aging of the population clinicians are increasingly confronted with elderly, often frail, patients with a DLBCL. While the very elderly patients, defined by the age of ≥75 year, are often excluded from clinical trials, only few prospective data in the rituximab-era are available on the outcome in this patient cohort. To asses efficacy, tolerability and safety of standard intensive rituximab-containing therapy in those patients, a descriptive population-based, cohort study was performed. Detailed information on treatment, toxicity and outcome in all patients aged ≥75 year, diagnosed with DLBCL in the Dutch province Friesland was prospectively gathered and analyzed. Methods Since 2005 all patients diagnosed with a haematological malignancy in Friesland, a province with 600.000 inhabitants, are prospectively registered and followed in a population-based registry, the HemobaseR. For this analysis data of all patients aged ≥ 75 years with a newly diagnosed DLBCL over a period of six years were retrieved from the HemobaseR. Data of clinical characteristics, treatment and its adaptations, treatment-related toxicity and outcome were obtained. Treatment modality was divided in four groups: R-CHOP chemotherapy, other rituximab-containing therapy, palliative radiotherapy and only supportive care. Follow-up was completed until 31 December 2011. Cox Proportional hazards model was used for identifying significant prognostic risk factors. Kaplan-Meier curves for each group were evaluated by a logrank test. Results From 2005 until the end of 2011 103 patients aged ≥ 75 years were diagnosed with a DLBCL. The median age was 81 years (range 75 – 96) with a slight female predominance (57%). The median observation period was 13 months (range 1 – 78) and 31 months for those still alive at time of evaluation.19 patients (19%) had stage 1 disease; in 74 patients (71%) advanced disease (stage 2–4) was reported and in 10 patients (10%) staging was incomplete. In 84 patients (81%) an age adjusted International Prognostic Index was calculated, 43% of them had an aaIPI of 2 or 3. Of the patients with stage 1 disease, 13 (68%) received three courses of R-CHOP and radiotherapy with a curative intent. Eight (62%) of them did complete this therapy. Of the other 84 patients 57 (68%) received R-CHOP chemotherapy with a curative intent. 31 (54%) of them completed at least six cycles. Ultimately 39 patients (56%), who did start with a standard therapy regimen, completed their treatment. A complete remission was achieved in 30 patients (77%). In the remaining group 10 patients (10%) received alternative, suboptimal, rituximab-based chemotherapy, 9 (9%) of patients was treated with radiotherapy. In 12 patients (11%) only supportive care was given. Severe toxicity (grade 3–4), occurred in 66% of all patients treated with rituximab-containing chemotherapy. Toxicity grade 3–4 was the main reason for receiving less then six cycles of chemotherapy (44%). Ten toxicity-related deaths (13%) were observed in patients treated with R-CHOP, with eight deaths due to infectious complications. The two-year survival was 67% for the elderly who completed chemotherapy, 25% for those treated with incomplete or inferior chemotherapy regimens and 20% for those receiving palliative radiotherapy or supportive care. In a multivariate analysis completing therapy and not age was significantly associated with a better survival (p