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Male sex is associated with unfavourable pharmacokinetics and prognosis in elderly patients with diffuse large B-cell lymphoma (DLBCL). We investigated higher rituximab doses for elderly male DLBCL patients. Elderly patients (61-80years) received 6 cycles CHOP-14 (cyclophosphamide, doxorubicin, vincristine and prednisone at 14-day intervals) and were randomized to 8 cycles rituximab (males 500 mg/m(2), females 375 mg/m(2)) every 2 weeks or according to an upfront dose-dense schedule. In 268 (120 females, 148 males) no difference between the standard and the upfront dose-dense rituximab schedule was found (3-year PFS 72% vs. 74%;OS 74% vs. 77%;P=0.651). The 500mg/m(2) dose of rituximab for male patients was associated with serum levels and exposure times slightly better than in females and a male/female hazard ratio of 0.9 for progression-free survival (PFS) and 0.8 for overall survival. For elderly males, 500 mg/m(2) was not more toxic than 375 mg/m(2) rituximab, but improved PFS by 32.5% (P=0.039), with a trend for a (30%) better overall survival (P=0.076) in a planned subgroup analysis adjusting for International Prognostic Index risk factors. We conclude that the higher rituximab dose for elderly male patients abrogated the adverse prognosis of male sex without increasing toxicity. In the era of personalized medicine, sex-specific pharmacokinetics and toxicities should be investigated for all drugs where these parameters impact on outcome.

We characterized genome alterations in 1255 clinically annotated lung tumors of all histological subgroups to identify genetically defined and clinically relevant subtypes. More than 55% of all cases had at least one oncogenic genome alteration potentially amenable to specific therapeutic intervention, including several personalized treatment approaches that are already in clinical evaluation. Marked differences in the pattern of genomic alterations existed between and within histological subtypes, thus challenging the original histomorphological diagnosis. Immunohistochemical studies confirmed many of these reassigned subtypes. The reassignment eliminated almost all cases of large cell carcinomas, some of which had therapeutically relevant alterations. Prospective testing of our genomics-based diagnostic algorithm in 5145 lung cancer patients enabled a genome-based diagnosis in 3863 (75%) patients, confirmed the feasibility of rational reassignments of large cell lung cancer, and led to improvement in overall survival in patients with EGFR-mutant or ALK-rearranged cancers. Thus, our findings provide support for broad implementation of genome-based diagnosis of lung cancer.

The therapeutic benefit of G-CSF in the treatment of acute lymphoblastic leukemia has been well established. G-CSF has been used to shorten neutropenia induced by conventional dose cytotoxic chemotherapy and allogeneic bone marrow transplantation. Recently autologous peripheral blood progenitor cell transplantation has been explored to treat high-risk ALL. Several in vitro studies suggest that subpopulations of lymphoblasts express G-CSF receptors. Furthermore, enhanced growth of Ph+ ALL cells expressing myeloid antigens stimulated by G-CSF has been demonstrated in vitro. However, the clinical relevance of these findings has been questioned. We report a patient with my+Ph+ALL in whom the administration of G-CSF after high-dose Cytarabin and Mitoxantrone led to a significant mobilization of leukemic cells and contamination of the stem cell harvest during cytologic marrow remission. Am. J. Hematol. 58:330–333, 1998. © 1998 Wiley-Liss, Inc.

Little is known about the use of first-line chemotherapy in clinical practice in patients with advanced esophagogastric adenocarcinoma, and no data have been published regarding potential obstacles for the implementation of molecular testing for targeted agents in this patient group. Here, we sought to evaluate factors influencing treatment decisions with special focus on the implementation of HER2 testing during the first year after trastuzumab market approval in Germany.A total of 754 patients undergoing treatment decisions for palliative first-line therapy in 2010 were documented using Therapiemonitor(®). Drug use and intensity of first-line treatment were analyzed. Data on HER2 testing and test algorithm are described, and variables influencing HER2 testing were selected using bivariate analysis. Significant factors were included in a multivariate logistic regression analysis.Compared with previous years, treatment intensity has further increased. The use of chemotherapy triplets rose from 10.1 % in 2006 to 60.3 % in 2010. In 2010, 49.1 % of patients were tested for HER2 and in 52.2 % of these patients the currently proposed test algorithm was used. Using multivariate logistic regression analysis age ≥67 years and "initiating institution: practice" were found to negatively impact the likelihood of HER2 testing, while treatment goal "prevention of progression", multiple metastases and a Karnofsky status80 % showed positive correlation with HER2 testing.The tendency to use more intensive first-line chemotherapy regimens in patients with advanced esophagogastric adenocarcinoma continued in 2010. Only a minority of patients had an access to the appropriate molecular diagnostics and therefore to treatment with trastuzumab. The access was limited due to the preselection following individual, clinical and institutional factors.

Multiple myeloma (MM) is a clonal plasma cell disorder frequently accompanied by hematopoietic impairment. We show that hematopoietic stem and progenitor cells (HSPCs), in particular megakaryocyte-erythrocyte progenitors, are diminished in the BM of MM patients. Genomic profiling of HSPC subsets revealed deregulations of signaling cascades, most notably TGFβ signaling, and pathways involved in cytoskeletal organization, migration, adhesion, and cell-cycle regulation in the patients. Functionally, proliferation, colony formation, and long-term self-renewal were impaired as a consequence of activated TGFβ signaling. In accordance, TGFβ levels in the BM extracellular fluid were elevated and mesenchymal stromal cells (MSCs) had a reduced capacity to support long-term hematopoiesis of HSPCs that completely recovered on blockade of TGFβ signaling. Furthermore, we found defective actin assembly and down-regulation of the adhesion receptor CD44 in MM HSPCs functionally reflected by impaired migration and adhesion. Still, transplantation into myeloma-free NOG mice revealed even enhanced engraftment and normal differentiation capacities of MM HSPCs, which underlines that functional impairment of HSPCs depends on MM-related microenvironmental cues and is reversible. Taken together, these data implicate that hematopoietic suppression in MM emerges from the HSPCs as a result of MM-related microenvironmental alterations.

For patients with acute myeloid leukemia refractory to intensive chemotherapy prognosis is very poor and treatment options are limited. 5-Azacytidine, a demethylating drug, is effective in the treatment of myelodysplastic syndromes when administered at a low-dose, subcutaneously. We report a case of a patient with AML refractory to induction chemotherapy as well as to two high-dose salvage regimens. The patient achieved CR through monotherapy with low-dose azacytidine.

Thalidomide is thought to have anti-angiogenic and immunomodulatory properties, including suppression of tumor necrosis factor-alpha, effects on interleukins and interferons, down-regulation of some cell adhesion molecules, and changes in the proportion of lymphocyte subsets. It is unclear whether the clinical response to thalidomide in patients with multiple myeloma (MM), idiopathic myelofibrosis (IM), and myelodysplastic syndromes (MDS) is related to its ability to inhibit angiogenesis or its immunomodulatory effects. We examined the effect of thalidomide on T-lymphocyte subsets in 18 patients with MDS, 6 patients with MM, 4 patients with IM, and 3 patients with angioimmunoblastic lymphoma (AILD). These patients had either a relapse or progressive disease following cytotoxic chemotherapy including high-dose chemotherapy with autologous stem cell support. Thalidomide was first administered at 100 mg/day p.o. and increased to 400 mg/day. T-lymphocyte subsets (CD4+, CD8+) were measured by fluorescence-activated cell sorter (FACS) before and during treatment with thalidomide. Twenty-six of 31 patients responded to thalidomide, most of them achieving partial remission. The median concentration of CD4+ cells was 443/microl, the median of CD8+ cells was 359/microl (CD3 992/microl). In our cohort, no significant changes in absolute numbers or proportions of CD3+ (P = 0.12), CD4+ (P = 0.668), or CD8+ (P = 0.143) cells were observed following the treatment with thalidomide. Although the CD4/CD8 ratio declined from 1.6 to 1.0 during 3 months of thalidomide treatment, this had no statistical significance (P = 0.1). Our findings show that an effect of thalidomide on the T lymphocytes studied is unlikely to be of major importance for the clinical effects.

Detection of minimal residual disease (MRD) has helped to improve the treatment of patients with leukemia. At present MRD testing in patients with multiple myeloma (MM) is not applied as a standard diagnostic or prognostic method.Immunoglobulin heavy chain (IgH) polymerase chain reaction (PCR) using patient-specific TaqMan probes together with LightCycler technology was performed to quantify minimal residual disease in MM. Relative levels of clonotypic cells were assessed as IgH/2beta-actin ratios with a sensitivity of 10(-4) to 10(-5).Following stem cell transplantation, a significant reduction of clonotypic cells was observed in bone marrow (BM) and peripheral blood (PB) samples of 11 patients, comparing pre-treatment values with those of best response (median: 13% to 0.09% and 0.03% to 0%, respectively). In 5 patients with ongoing clinical remission IgH/2beta-actin ratios remained stable at a low level, while in 6 patients an increase to 2% in BM and 0.4% in PB was associated with progression of the disease. In 4 of these 6 patients the increase of clonotypic cells in PB was detectable a median of 3 months (range: 0.5-6) before relapse. Furthermore, time-to-progression of patients with pre-transplantation IgH/2b-actin ratios0.03% in BM was significantly shorter than that of patients with lower MRD levels.MRD in patients with MM can be quantified reliably using TaqMan chemistry adapted to the LightCycler system. Residual tumor cell levels before transplantation as well as results of sequential molecular monitoring are predictive of relapse.

We report on our experience in the use of transdermal fentanyl in management of acute pain due to mucositis WHO-grade IV during high-dose chemotherapy (HDC) and autologous stem cell support (APBSCT). Between 8/96 and 12/98 74 patients received HDC and PBSCT for progressive disease or relapse of non-Hodgkin's lymphoma (n=32), multiple myeloma (n=37), Hodgkin's lymphoma (n=5). All patients suffered from mucositis WHO-grade IV with a need for continuous pain management. Instead of pethidine i.v. fentanyl TTS was used. Sufficient analgesia was achieved mostly with a dose of 50 microg/h. There was no need of supplementary analgesia. Relevant fentanyl-associated side effects were not seen. Patient compliance and acceptance were excellent. The results suggest that transdermal fentanyl is reliable in pain management of chemotherapy-associated mucositis grade IV.

Abstract 2898 Multiple myeloma (MM) patients often suffer from hematopoietic impairment already at the time of diagnosis with anemia as the prevailing symptom. Given the overt affection of the bone marrow in MM patients by the invasion of malignant plasma cells, we hypothesized that hematopoietic insufficiency in these patients may originate from a functional impairment of hematopoietic stem and progenitor cells. Quantitative analysis of BM CD34+ HSPC cell subsets from MM patients and age-matched healthy donors showed a significant decline of all HSPC subsets including hematopoietic stem cells, common myeloid and lymphoid progenitors, granulocyte-macrophage progenitors and megakaryocyte-erythrocyte progenitors in MM patients. The greatest diminution was observed in megakaryocyte-erythrocyte progenitors (MEP) which were 4.9-fold reduced in comparison to healthy donors. Transcriptional analyses of CD34+ HSPC subsets revealed a significant deregulation of signaling pathways that was particularly striking for TGF beta signaling and suggested increased activation of this signaling pathway. Immunhistochemical staining of phosphorylated smad2, the downstream mediator of TGF receptor I kinase activation, in bone marrow sections and immunoblotting of purified CD34+ HSPC of MM patients confirmed the overactivation of TGF beta signaling. On a functional level, we observed significantly reduced long-term self-renewal and clonogenic growth, particularly of the erythroid precursors BFU-E and CFU-E, in CD34+ HSPC of MM patients which could be restored by inhibition of TGF beta signaling. Proliferation and cell cycle analyses revealed a significantly decreased proliferation activity in CD34+ HSPC and, particularly, MEP. Again, this was reversible after inhibition of TGF beta signaling. In addition, the transcriptional analyses showed disturbance of pathways involved in the adhesion and migration of HSPC and the gene encoding for the principal hyaluronan receptor CD44 throughout the HSPC subsets. This was corroborated by immunofluorescence imaging of CD44 on HSPC subsets showing a marked downregulation in the patients' cells. In line, the adhesion of CD34+ HSPC subsets to hyaluronan and their migration towards SDF-1 was significantly inhibited. Subsequent xenotransplantation of CD34+ HSPC from MM patients and healthy donors into myeloma-free recipients revealed even increased long-term engraftment of CD34+ HSPC obtained from MM patients and normal differentiation capacities suggesting that the observed functional alterations in fact depend on the MM-related bone marrow microenvironment. Our data show that hematopoietic impairment in patients with multiple myeloma originates, at least in part, from functional alterations of hematopoietic stem and progenitor cells. These alterations seem to depend on the disease-related changes of the bone marrow microenvironment. Currently, experiments are underway to elucidate in more detail the role of the microenvironment and the responsible structures for the impairment of HSPC in MM patients. These data will be presented. Disclosures: Kobbe: Celgene: Consultancy, Research Funding; Ortho Biotec: Consultancy.

Abstract 2393 Background: Myelodysplastic syndromes (MDS) represent a heterogeneous group of hematopoietic stem cell disorders and research in this field has mainly focused on hematopoietic stem and progenitor cells (HSPC). Still, recent data from mouse models indicate that the bone marrow (BM) microenvironment might be involved in the pathogenesis MDS (Raaijmakers et al., 2010). The role of mesenchymal stromal cells (MSC) in particular as a key component of the BM microenvironment remains elusive in human MDS and data so far are controversial. Design/Methods: We therefore investigated MSC and immunomagnetically enriched CD34+ HSPC from BM of 42 untreated patients (pts) with MDS (12 RCMD, 12 RAEB, 12 sAML, 3 del5q, 1 CMML-1, 1 MDS hypocellular, 1 MDS unclassifiable according to WHO) and age-matched healthy controls (HC, n=13). MSC were examined with regard to growth kinetics, morphology and differential potential after isolation and expansion according standard procedures in line with the international consensus criteria (Dominici et al., 2006). Furthermore corresponding receptor-ligand pairs on MSC and CD34+ cells (Kitlg/c-kit; CXCL12/CXCR4; Jagged1/Notch1; Angpt1-1/Tie-2; ICAM1/LFA-1) were investigated by RT-PCR. Results: In MDS, the colony forming activity (CFU-F) of MSC was significantly reduced in comparison to HC (median number of colonies per 1×107MNC in MDS: 8, range 2–74 vs. HC: 175, 10–646, p=0.003) and this was also true when looking at the different subtypes (RCMD median: 16, p=0.04; RAEB median: 8, p=0.31; sAML median: 26, p=0.02). According to this, MSC from pts with RCMD and del5q could only be maintained in culture for a lower number of passages (median number of passages: MDS 3 passages, range 1–15; HD 14 passages, range 8–15, p=0.01), had a lower number of cumulative population doublings (CPD) and needed a longer timer to reach equivalent CPD (MDS median: 18,16 CPD, HD median: 33,68 CPD, p=0,0059). All types of MDS-MSC showed an abnormal morphology, while an impaired osteogenic differentiation potential was exclusively observed in pts with RCMD. These findings of an altered morphology together with a diminished growth and differentiation potential prompted us to test, whether the interaction between MSC and CD34+ HSPC in BM of pts with MDS was also disturbed. On the MDS-MSC, we found a significant lower expression of Angpt1 in pts with RAEB (3.5-fold, p=0.01) and del5q (4.9-fold, p=0.009) compared to HD. The expression of CXCL12 (2.5-fold, p=0.057) and jagged1 was reduced in trend in MSC from pts with MDS, while no differences were observed with regard to the expression of kitlg and ICAM1. When looking on CD34+ cells, we found a significantly reduced expression of CXCR4 (RCMD 2.5-fold, p=0.02; RAEB 2.46-fold, p=0.02), notch1 (RCMD 6-fold, p=0.04) and Tie-2 (RAEB 5.91-fold, p=0.02) in pts with MDS, while LFA-1 was overexpressed in pts with RAEB (2.6-fold, p=0.036). Conclusion: Taken together, our data indicate that MSC from pts with MDS are structurally altered and that the crosstalk between CD34+ HSPC and MSC in the BM microenvironment of pts with MDS might be deregulated as a result of an abnormal expression of relevant receptor-ligand pairs. Ongoing research is required to corroborate these findings and to definitely address their functional relevance for the pathogenesis of MDS. Disclosures: No relevant conflicts of interest to declare.