Abstract: There is a higher incidence of hematological clonal stem cell disorders in elderly persons. Age-related alterations of hematopoietic stem and progenitor cells (HSC) may represent one factor underlying this observation. However, the molecular changes related to stem cell aging are largely unknown. Therefore, we scrutinized gene expression patterns of HSC from umbilical cord blood (CB) as well as bone marrow from young (BM-Y, mean age 32,8, SD 12,4) and old (BM-O, mean age 88,8, SD 4,4) healthy donors. CD34+ HSC were isolated via immuno-magnetic separation and CD34+ purity was evaluated by FACS analysis. Thereafter we performed cDNA array analyses on a first set of samples (n=18). We found that the BCL2-interacting killer gene (BIK) and the gene encoding KU Antigen 70kD (KU70) show age-related mRNA expression levels. BIK is a proapoptotic gene and its expression was positively correlated with donor’s age, i.e. lowest in CB, 1.8-fold higher in BM-Y and 4.2-fold higher in BM-O. KU70 is a DNA repair gene and part of the DNA dependent protein kinase (DNA-PK). Its expression was negatively correlated with donor’s age showing highest expression levels in CB, 2.2-fold lower levels in BM-Y and 5.3-fold lower levels in BM-O. These findings were confirmed with a second set of samples (n=16) by means of quantitative RT-PCR. Elucidation of age-dependent molecular alteration in healthy HSC might facilitate a better understanding of hematological malignancies in elderly persons.

Abstract: Background: Relapse of aggressive B-cell as well as peripheral T-cell lymphoma (PTLC) has a very poor prognosis, especially in pts ineligible for high-dose chemotherapy. Nivolumab, a human anti-PD1 antibody has the potential to increase rituximab-mediated effector mechanisms and to target the microenvironment in Non-Hodgkin lymphoma. Addition of nivolumab might also increase the efficacy of conventional chemotherapy. Therefore, the NIVEAU trial is testing a common conventional chemotherapy regimen (R)-GemOx vs. Nivolumab plus (R)-GemOx (ClinicalTrials.gov Identifier:NCT03366272). Design and study population: This is an ongoing joint international, multicenter, randomized, open label study proceeded by a safety run-in phase conducted by several European cooperative study groups (LYSA, HOVON, PLRG, KLS, AGMT, GLA). Key eligibility criteria include: first relapse or progression of an aggressive lymphoma (B-cell as well as PTCL), ineligibility for HDT (defined as 〉 65 years of age or older than 18 years if HCT-CI score 〉 2 or pts who underwent prior autologous stem cell transplantation and are not eligible for allogeneic stem cell transplantation), only one prior chemotherapy regimen including an anthracycline and rituximab (R) in case of B-cell lymphoma. Statistical methods: The primary endpoint is 1-year PFS rate. We aim to demonstrate an improvement from 27% to 42% (i.e. a hazard ratio of 0.66), importantly in B-cell lymphoma only. The two-sided question will be answered with an error probability of alpha= 5% (two sided) and a power of 80%. Therefore, it will be necessary to analyze 292 pts. A drop-out rate of 5% of pts results in a sample size of 310 pts with B-cell lymphoma to be randomized. In parallel a maximum of 78 pts with PTCL will be included and randomized without statistical assumption. Based on the results and possible further increasing scientific knowledge an estimation will be done whether an improvement of prognosis of PTCL can be expected. Potentially an adapted statistical hypothesis will be defined and the trial amended to recruit the appropriate sample size of PTCL. Analysis: A non-randomized safety run-in phase with 16 pts (10 DLBCL/6 PTCL) was performed in 2018. A 2nd safety analysis will be done after randomization of 30 pts to the experimental arm to allow for comparative description of toxicities between the arms. An interim analysis of efficacy will be performed including the first 180 patients from the B-cell cohort. The final analysis of the primary and secondary endpoint will be performed two years after recruitment of the last pt with B-cell lymphoma. A comprehensive translational research program will be performed on paired samples of primary diagnosis and relapse (NGS targeted resequencing, gene expression profiling, comparative genomic hybridization), PBMC (flow cytometry), serum, plasma and cell-free DNA. Study treatment: (R)-GemOx is Gemcitabine 1000 mg/m2, d1, Oxaliplatin 100 mg/m2, d1, Rituximab 375 mg/m2 in case of B-cell lymphoma disease, repeated every 2 wks. Standard arm: eight cycles of (R)-GemOx. Experimental arm: eight cycles of nivolumab (3 mg/kg) plus (R)-GemOx in 2-wk intervals followed by additional 18 infusions of Nivolumab (3 mg/kg) in 2-wk intervals as consolidation or up to progression or unacceptable toxicity. Endpoints: The primary endpoint is 1-year PFS. Secondary endpoints represent efficacy (response rates, duration of response, progression rate, relapse rate, EFS, OS), toxicity (AEs, SAEs, treatment related deaths, secondary malignancies, long-term sequelae), protocol adherence (number of chemotherapy cycles, duration of chemotherapy cycles, cumulative dose and relative dose of gemcitabine, oxaliplatin, rituximab, nivolumab), quality of life (assessed by 5Q-5D-5L) and outcome according to biology. Conclusion: The NIVEAU-study represents a trial with a clear phase 3 design aiming to improve outcome in pts. with relapsed aggressive B-cell lymphoma ineligible for HDT. Additive recruitment and randomization of PTCL allows to adapt and define a precise statistical assumption in future, depending on interim-results, upcoming knowledge and recruitment. The approach allows to provide phase 3 evidence also in very rare disease entities. Disclosures Held: Bristol-Myers Squibb: Consultancy, Other: Travel support, Research Funding; Roche: Consultancy, Other: Travel support, Research Funding; Amgen: Research Funding; Acrotech: Research Funding; MSD: Consultancy. Houot:Bristol Myers Squibb: Honoraria; Merck Sharp Dohme: Honoraria. André:Takeda: Membership on an entity's Board of Directors or advisory committees; Bristol-Myers-Squibb: Membership on an entity's Board of Directors or advisory committees; Karyopharm: Membership on an entity's Board of Directors or advisory committees; Gilead: Membership on an entity's Board of Directors or advisory committees, Other: Travel grants; Novartis: Membership on an entity's Board of Directors or advisory committees, Research Funding; Seattle Genetics: Membership on an entity's Board of Directors or advisory committees; Abbvie: Membership on an entity's Board of Directors or advisory committees; Roche: Other: Travel grants, Research Funding; Amgen: Other: Travel grants, Research Funding; Johnson & Johnson: Research Funding; Takeda Millenium: Research Funding; Chugai: Research Funding; Celgene: Other: Travel grants, Research Funding. Jaeger:Novartis, Roche, Sandoz: Consultancy; AbbVie, Celgene, Gilead, Novartis, Roche, Takeda Millennium: Research Funding; Amgen, AbbVie, Celgene, Eisai, Gilead, Janssen, Novartis, Roche, Takeda Millennium, MSD, BMS, Sanofi: Honoraria; Celgene, Roche, Janssen, Gilead, Novartis, MSD, AbbVie, Sanofi: Membership on an entity's Board of Directors or advisory committees. Trneny:Roche: Consultancy, Honoraria; Bristol-Myers Squibb: Consultancy, Honoraria; Amgen: Consultancy, Honoraria; Abbvie: Consultancy, Honoraria; Incyte: Consultancy, Honoraria; Gilead sciences: Consultancy, Honoraria; Takeda: Consultancy, Honoraria; Janssen: Consultancy, Honoraria; Morphosys: Consultancy, Honoraria. Maria:Janssen Cilag: Consultancy, Other: Travel support; Gilead Sciences: Other: Travel support, Research Funding; Abbvie: Consultancy, Other: Travel support; Celgene: Consultancy; Roche: Consultancy, Other: Travel support. Rosenwald:MorphoSys: Consultancy. Rymkiewicz:Roche: Other: Travel support. Tarte:Celgene: Consultancy, Research Funding; Roche: Consultancy, Research Funding. Poeschel:Abbvie: Other: Travel support; Amgen: Other: Travel support; Roche: Other: Travel support; Hexal: Speakers Bureau. Haioun:Amgen: Honoraria; Celgene: Honoraria; Gilead: Honoraria; Janssen: Honoraria; Novartis: Honoraria; F. Hoffmann-La Roche Ltd: Honoraria; Servier: Honoraria; Takeda: Honoraria; Miltenyi: Honoraria. OffLabel Disclosure: nivolumab, aggressive Non-Hodgkin Lymphoma

Abstract: Peripheral T-cell lymphoma is a rare heterogeneous disease with generally poor outcome. Known risk factors include the International Prognostic Index (IPI) and b2 microglobulin. However, there is little information on molecular risk factors. In this single center analysis we have prospectively studied the prognostic value of a clonal T-cell receptor rearrangement (TCR) determined by conventional PCR for the γTCR (sensitivity 1 in 102) from DNA from peripheral blood MNC. Thirty nine consecutive patients diagnosed between 1987 and 2006 were assessed for clonality at diagnosis. Patient characteristics: Median age 53 years (range 28–89). M:F = 29 vs. 10. Histological diagnosis included: PTCL-NOS 7 (18%); Large cell 1 (2.5%); Large + medium-sized 5 (13%); Pleomorphic 7 (18%); Small cell 3 (8%); Small+medium mixed cell 1 (2.5%); Lennert’s type 4 (10%); Hepatosplenic 3 (8%); Intestinal 4 (10%); Kimura’s disease: 1 (2.5%); AILD: 3 (8%). Assessment of γTCR showed monoclonality in 24 (62%) and polyclonality in 15 (38%) patients. Patients with a monoclonal TCR had higher clinical stages, higher LDH levels, higher IPI scores, and higher ß2 microglobulin levels. Clinical stage: CS I–III (clonal 42%; polyclonal 58%) vs. CS IV (clonal 93%; polyclonal 7%). LDH above 240 U/L (clonal 78%; polyclonal 22%). IPI 0,1 (clonal 44%; polyclonal 56%), IPI 2 (clonal 75%; polyclonal 25%), IPI 3 (clonal 87%; polyclonal 13%), IPI 4,5 (clonal 60%; 40%). ß2 microglobulin above 2 mg/L (clonal 68%; polyclonal 32%). Patients with a clonal TCR tended to be older (57 vs. 49 yrs.). No major differences were found for age and sex. Induction treatment consisted of polychemotherapy (CHOP-like=34), prednisone (1), radiation (1). 3 patients received no therapy. Nine patients received subsequent involved field radiation and 6 pts. had an autologous stem cell transplant. Significant differences were observed in terms of disease outcome: The presence of a clonal γTCR (n=24) was associated with low clinical remission rates: CR 38%, PR 21%, SD 8%, PD 33%, relapse rate 38%. Remission rates in patients with polyclonal rearrangements (n=15) were: CR 66%, PR 20%, SD 7%, PD 1%, relapse rate 47%. The estimated 3-year overall survival for the monoclonal group was 26% vs. 69% for the polyclonal group (P=0.01). Importantly, patients with CS I–III and a monoclonal γTCR rearrangement (n=10) had a similarly poor survival when compared to the monoclonal group with CS IV (n=14) (18 months vs. 12 months; P=0.23). This indicates that γTCR positivity is of particular predictive value in patients with CS I–III. The data from this unique cohort suggest that γTCR-PCR is a useful prognostic tool in patients with peripheral T-cell lymphoma. Figure Figure

Abstract: Maintenance therapy with the monoclonal anti-CD20 antibody Rituximab (R) improves clinical outcome in patients with follicular lymphoma in complete or partial remission (CR, PR) after induction therapy. However, optimal dosing schedule and duration of maintenance therapy are still under investigation. Commonly applied regimen include four doses of 375mg/m2 Rituximab once a week every 6 months, one dose every 3 months, or one dose every 2 months. Maintenance is usually given for 2 years. In addition to clinical trials, pharmacokinetic (PK) studies could help determining optimal dosing of Rituximab. PK values have been obtained during induction therapy in several trials and an association between drug trough levels and clinical effectiveness has been established (25μg/ml in responding patients). However, data on Rituximab PK during maintenance therapy are lacking. The Austrian Cooperative Study Group for Cancer Drug Therapy (AGMT) has conducted a phase II trial in 33 patients with follicular lymphoma in CS III/IV with an induction therapy with 6 cycles of Rituximab (375mg/m2 i.v., day 1), Fludarabine (30mg/m2 p.o., day 2–4)) and Mitoxantron (10mg/m2 i.v., day 1) every 4 weeks (R-FM) (NHL9). Thirty two patients obtaining a CR, CRu or PR received maintenance treatment with Rituximab 375mg/m2 every 2 months for 1 year. Pharmacokinetic data for Rituximab were obtained during induction and maintenance treatment in 16 of these patients. All patients converted from BCL2/IgH PCR positivity to negativity in peripheral blood after R-FM induction. PK serum concentrations during induction corresponded well to known data: A steady increase in pre- and post-dose levels was observed with increasing cycle number. Median, minimum and maximum Rituximab serum concentrations are given in the upper panel of Table 1: Table 1. Serum concentrations (μg/ml) during R-FM induction and two-monthly maintenance treatment with 375mg/m2 of Rituximab. R-FM Induction Cycle 1 2 4 6 (Q 4 weeks) min. med. max. min. med. max. min. med. max. min. med. max. Pre-dose N.A. & lt;0.5 N.A. & lt;0.5 16.8 44.8 25.3 56.6 93.2 43.2 66.5 208.3 Post-dose 44.8 161.4 236.7 146.4 204.6 300.5 176.6 218.4 346.2 196.3 249.5 370.2 Interval 2 months R-Maintenance Cycle 1 2 4 6 (Q 2 months) min. med. max. min. med. max. min. med. max. min. med. max. Pre-dose 13.8 41.5 113.7 6.2 31.0 72.8 14.8 35.6 261.2 13.7 26.7 56.0 Post-dose 136.6 237.9 364.6 198.0 235.9 350.7 214.2 235.0 280.9 185.1 249.2 271.1 The median interval between day 1 of the last R-FM induction cycle and the initial dose of R maintenance treatment was 2 months (range 1 – 3). PK values were obtained during maintenance cycles 1, 2, 4, and 6. The median interval from the first Rituximab maintenance infusion to the sixth infusion was 10.4 months (range 9.7 – 10.5). Table 1 (lower panel) shows the median, minimum and maximum drug levels before and after R maintenance infusions. These results indicate that (i) Median Rituximab levels 2 months after the last infusion of induction treatment are still higher than the supposedly active concentration of 25μg/ml; (ii) Rituximab target levels of over 25μg/ml are constantly maintained throughout one year of maintenance. (iii) Rituximab trough levels may still be low in some patients on a two monthly schedule. There was no difference in maintenance trough levels between CR and PR patients. So far, no relapses were observed. We conclude that infusion of Rituximab 375mg/m2 once every two month results in constant active serum levels in patients with follicular lymphoma in complete or partial remission. These data should help designing rational maintenance schedules for future clinical trials.

Abstract: Background: Aggressive hematological malignancies in relapsed/refractory setting bear a dire prognosis with low cure rates and short survival. Matching these patients to therapies is challenged by complexity due to spatial and temporal tumor evolution and incomplete understanding of genotype to phenotype correlations. Direct functional testing could address these impediments. The EXALT trial is an interventional, one-arm study designed to assess the clinical value of next generation functional drug screening (ngFDS). An interim analysis on 17 patients suggested a clinical benefit (Snijder et al., Lancet Hematol. 2017). Methods: We applied image-based ngFDS to quantify differential ex-vivo sensitivity of primary patient tumor cells to respective non-tumor cells towards 136 small molecule drugs, including EMA approved for any indication or experimental. We screened bone marrow, peripheral blood or lymph node material from 143 patients who suffered from late stage aggressive hematological malignancies (acute leukemias, aggressive B- and T-cell lymphomas) , discussed the results in a multidisciplinary tumor board and recommended treatments to physicians (A). The primary endpoint of this study was the percentage of patients reaching a PFS-ratio (PFS(ngFDS treatment)/PFS(previous treatment)) of ≥1.3 with an H0 hypothesis & lt; 15% patients. The secondary endpoint was overall response rate (ORR) defined as proportion of patients reaching complete remission (CR) or partial remission (PR). Additionally, we performed a post hoc analysis to evaluate the matching of ngFDS to drugs used in actual treatment (matching score of received treatment). Results: 56 (39%) patients were evaluable and treated according to ngFDS based recommendations. With 30 of 56 (54%) ngFDS guided patients experiencing a PFS ratio of ≥1.3, the primary study endpoint was reached. 11 patients (37%) had ongoing response at censoring date (B). The median follow-up was 718 days. The median number of days from sampling to treatment was 21 (range 4-77). The ngFDS treatment regimens consisted of a median of 2 drugs (range: 1-6). ORR was 55% for all evaluable ngFDS treated patients, 60% for the lymphoid subgroup and 41% for the myeloid subgroup. Patients on ngFDS guided treatment with performance status ECOG ≤ 1 had a median PFS of 207 days compared to a median PFS of 29 days for patients with higher ECOG (p & lt; 0.001, C). 24 of 39 (62%) patients with ECOG ≤ 1 had a PFS ratio of ≥1.3 (D). In disease specific subgroup analysis median PFS of T-cell lymphoma patients was 235 days versus 60 days for B-cell lymphoma patients (p = 0.018, E). Age (≤60 vs. & gt;60), sex, lineage (myeloid vs. lymphoid), number of previous treatment lines (≤2 vs. & gt;2), and clinical presentation (leukemia vs. lymphoma) did not have an impact on PFS of ngFDS guided treatment. Post hoc analysis including additional 17 non-ngFDS treated patients demonstrated that only patients receiving treatment with a positive ngFDS matching score demonstrated clinical benefit (HR: 0.53, p=0.005; vs. HR: 1.4, p=0.4). ngFDS matched treatments resulted in higher PFS for patients with tumor samples that had a cancer cell fraction of 10-50% in comparison to patients with samples of lower or higher cancer cell percentage (HR:0.35, p=0.01). Conclusion: ngFDS could be integrated in the routine clinical work flow. ngFDS guided treatments led to high rates of PFS prolongation compared to previous treatments of individual patients. ngFDS guided treatment is feasible and effective in patients with late stage aggressive hematological malignancies. These results prompted a prospective randomized trial comparing treatment guidance based on ngFDS or comprehensive genomic profiling or physician's choice (EXALT-2 trial, NCT04470947). Figure Disclosures Vladimer: Allcyte GmbH: Current Employment, Current equity holder in private company, Other: Founder. Jaeger:Karyopharm: Honoraria; Amgen: Honoraria; Gilead: Honoraria, Research Funding; BMS/Celgene: Consultancy, Honoraria, Research Funding; True North: Honoraria, Research Funding; Miltenyi: Consultancy, Honoraria; CDR Life AG: Consultancy, Research Funding; F. Hoffmann-La Roche: Honoraria, Research Funding; Infinity: Honoraria; Takeda: Honoraria; Novartis: Consultancy, Honoraria, Research Funding; AbbVie: Honoraria. Krall:Allcyte GmbH: Current Employment, Current equity holder in private company, Other: Founder. Valent:Allcyte GmbH: Research Funding; Cellgene: Honoraria, Research Funding; Pfizer: Honoraria. Wolf:Celgene: Honoraria, Research Funding. Zielinski:MSD: Consultancy, Honoraria, Speakers Bureau; Roche: Consultancy, Honoraria, Speakers Bureau; Novartis: Consultancy, Honoraria, Speakers Bureau; BMS: Consultancy, Honoraria, Speakers Bureau; Imugene: Consultancy, Honoraria, Speakers Bureau; Ariad: Consultancy, Honoraria, Speakers Bureau; Pfizer: Consultancy, Honoraria, Speakers Bureau; Merrimack: Consultancy, Honoraria, Speakers Bureau; Merck KGaA: Consultancy, Honoraria, Speakers Bureau; Fibrogen: Consultancy, Honoraria, Speakers Bureau; AstraZeneca: Consultancy, Honoraria, Speakers Bureau; Tesaro: Consultancy, Honoraria, Speakers Bureau; Gilead: Consultancy, Honoraria, Speakers Bureau; Servier: Consultancy, Honoraria, Speakers Bureau; Shire: Consultancy, Honoraria, Speakers Bureau; Eli Lilly: Consultancy, Honoraria, Speakers Bureau; Athenex: Consultancy, Honoraria, Speakers Bureau. Superti-Furga:Allcyte GmbH: Current equity holder in private company, Other: Founder. Snijder:Allcyte GmbH: Current equity holder in private company, Other: Founder. Staber:Roche: Consultancy, Honoraria, Research Funding; Astra Zeneca: Consultancy, Honoraria; Celgene/ BMS: Consultancy, Honoraria; msd: Consultancy, Honoraria; Takeda: Consultancy, Honoraria; Janssen: Consultancy, Honoraria; Gilead: Consultancy, Honoraria; AbbVie: Consultancy, Honoraria.

Abstract: Although HIV-associated multicentric Castleman disease (HIV-MCD) is not classified as an AIDS-defining illness, mortality is high and progression to lymphoma occurs frequently. At present, there is no widely accepted recommendation for the treatment of HIV-MCD. In this retrospective (1998-2010), multicentric analysis of 52 histologically proven cases, outcome was analyzed with respect to the use of different MCD therapies and potential prognostic factors. After a mean follow-up of 2.26 years, 19 of 52 patients died. Median estimated overall survival (OS) was 6.2 years. Potential risk factors, such as older age, previous AIDS, or lower CD4 T cells had no impact on OS. Treatment was heterogeneous, consisting of cytostatic and/or antiviral agents, rituximab, or combinations of these modalities. There were marked differences in the outcome when patients were grouped according to MCD treatment. Patients receiving rituximab-based regimens had higher complete remission rates than patients receiving chemotherapy only. The mean estimated OS in patients receiving rituximab alone or in combination with cytostatic agents was not reached, compared with 5.1 years (P = .03). Clinical outcome and overall survival of HIV-MCD have markedly improved with rituximab-based therapies, considering rituximab-based therapies (with or without cytostatic agents) to be among the preferred first-line options in patients with HIV-MCD.

Abstract: We have previously shown that NOTCH2 signaling is involved in the overexpression of CD23 in B-cell chronic lymphocytic leukemia (B-CLL) cells (Hubmann et al., BLOOD 2002 May 15;99(10):3742–7). NOTCH2 plays a determining role in the development/homeostasis of CD5+ B1 B-cells and of the related marginal zone (MZ) B2 B-cells, suggesting a potential role for NOTCH2 in B-CLL leukemogenesis. Using electrophoretic mobility shift assays (EMSA) we demonstrate that freshly isolated B-CLL patient samples (n=30) express an activated form of nuclear NOTCH2 (N2IC) irrespective of their prognostic marker profile (ie. IgVH mutational status, CD38 surface expression, cytogenetics). Although the majority of cultured B-CLL samples lose their N2IC activity within one day, DNA-bound N2IC complexes could be maintained by low concentrations of the PKC-stimulating phorbol esther PMA (1ng/ml). This was accompanied by the upregulation of CD23. The effect of PMA on N2IC activation and CD23 expression was abrogated by the PKC-δ inhibitor Rottlerin. Since wild type NOTCH2 signaling is regulated through binding to its ligand followed by γ-secretase mediated cleavage and release of the intracellular domain (N2IC), we induced NOTCH2 signaling by PMA in the presence or absence of the γ-secretase inhibitors (GSI) DAPT and compound E. Results demonstrated that the PMA-induced NOTCH2 activity is resistant to GSI treatment in 24 out of 30 B-CLL cases (80%). This suggests that the leukemic cells from the majority of B-CLL patients express an activated form of N2IC which is independent from γ-secretase cleavage and, thus, do not reqire NOTCH2 ligands for signaling. In conclusion, the results suggest that PKC-δ is involved in the activation/nuclear translocation of N2IC in B-CLL cells. The data may also suggest that deregulated NOTCH2 signaling is an early step in the development of B-CLL and might be critically involved in the aberrant response of the malignant clone to cell fate modulating stimuli acting through PKC.

Abstract: We have recently shown that NOTCH2 signaling is involved in the overexpression of CD23 in B-cell chronic lymphocytic leukemia (B-CLL) cells (Hubmann et al., BLOOD 2002). There is an increasing evidence that NOTCH2 plays a determining role in the development/homeostasis of self-reactive CD5+ B-cells, suggesting a potential function of NOTCH2 in B-cell leukemogenesis. Here we study the regulation of NOTCH2 signaling in B-CLL cells and its possible function in B-lymphocytes using NOTCH2 transduced BL41 cells as a model system. Cultured B-CLL samples (n=30) lose their nuclear NOTCH2 (N2IC) activity within one day as demonstrated by electrophoretic mobility shift assays (EMSA). However, DNA-bound NOTCH2 complexes could be maintained in culture by exposure to the phorbolester TPA and is accompained by increased cell viability. The effect of TPA is prevented by the PKC-δ inhibitor Rottlerin indicating that PKC-δ is involved in the regulation of NOTCH2 signaling in B-CLL cells. The activity of N2IC in the leukemic cells appeared to be resistant to the γ-secretase inhibitors DAPT and compound E, two substances known to block ligand mediated release of the NOTCH2 intracellular domain (N2IC). Since B-CLL cells are locked in an anergic state, we next asked whether NOTCH2 modulates B-cell receptor (BCR) signaling and found that retrovirally transduced N2IC rescues the B-cell line BL41 from surface immunoglobulin M (sIgM) mediated apoptosis, a mechanism proposed to prevent the uncontrolled expansion of self-reactive CD5+ B-cells. In summary, our data suggest that B-CLL cells express an activated form of NOTCH2 which might be involved in the protection of the malignant clone from peripheral negative selection.

Abstract: Conventional anthracyclines may have severe dose-limiting side effects in patients with pre-existing cardiac comorbidity, particularly if they are elderly. In routine practice, this will lead to dose reductions or preclude the use of anthracyclines. This may result in lower remission and shorter lymphoma-free or overall survival rates. Non-pegylated liposome-encapsulated doxorubicin (Myocet®) in combination with Rituximab, cyclophosphamide, vincristine and prednisone (R-COMP) has been shown to be effective with a low cardiotoxicity profile in patients with diffuse large B-cell lymphoma (DLBCL) (Rigacci et al., Hematol Oncol.2007;25:198–203). We have treated 37 lymphoma patients with pre-existing cardiac disorders or elderly patients with reduced general condition who were considered ineligible or high risk for anthracycline containing therapy. Thirty seven patients with various histologies were included: 20 DLBCL, 9 T-/NK cell lymphomas, 3 follicular lymphomas, 2 post-transplant lymphoproliferative disease (PTLD), 2 chronic lymphocytic leukemia (CLL) and 1 multiple myeloma (MM). Reasons for the use of liposome-encapsulated doxorubicin were: pre-existing cardiac disorders (N=19) including myocardial infarction, cardiomyopathy, reduced left ventricular ejection fraction LVEF (N=6), elevated B-type natriuretic peptide (BNP) levels (N=10, median 680 pg/ml), aortic valve problems, or heart transplantation; older age (N=12; median age 78 years); previous therapy with anthracyclines (N=3); other comorbity or reduced general condition (N=4). Twenty-nine patients (78%) were previously untreated. The following combination regimens were given: R-COMP (N=25), COMP (N=11), and BMD (N=1). The median number of Myocet® containing cycles administered was 5. For various reasons, liposome encapsulated doxorubicin was reduced to a dose of less than 50mg/m2 in 19 patients. High remission rates were observed for this poor risk population: Complete remission rates were 70% (14/20) for DLBCL and 57% (4/7 evaluable patients) for T-/NK cell lymphomas. 31 patients (83%) are still alive at 2–9 months after the end of therapy. Five patients died from progressive disease, one of unknown reasons. Thirteen patients with DLBCL are in continuous CR 3 months after the end of therapy (range 1–8 months). No major cardiac or gastrointestinal toxicity was observed. Of note, paravasation of liposome-encapsulated doxorubicin occurred in 2 patients, but resulted in mild inflammation only without tissue damage. Hematologic toxicity was comparable to that of conventional anthracycline containing regimens: CTC Grade 3 or 4 toxicity for leukocytopenia/neutropenia occurred in 22 patients (59%). Nineteen patients (51%) received G-CSF for primary or secondary prophylaxis in at least one cycle of treatment. We conclude that liposome-encapsulated doxorubicine is highly active in combination chemotherapy for B-cell lymphomas with low cardiac toxicity in patients with pre-existing cardiac disorders or older age. Moreover, we herewith report for the first time efficacy in T/NK-cell lymphomas in this high-risk population. A randomized study evaluating cardiac effects of substitution of conventional doxorubicin by liposome-encapsulated doxorubicin in combination chemotherapy (R-CHOP vs. R-COMP) is currently being conducted by the Austrian Cooperative Group on Cancer Drug Therapy (AGMT).

Abstract: Inhibition of apoptosis and long survival leads to accumulation of the leukemic cells in B cell chronic lymphocytic leukemia (B-CLL). This could be due to activation of anti-apoptotic cascades in CLL cells through interaction with their lymphoid microenvironment. Therefore, we investigated the role of tumor microenvironment in prolongation of survival of B-CLL cells and activation of the potent anti-apoptotic PI3-K/Akt pathway. Stromal fibroblasts of bone marrow (BMFs), spleen (SF) and lymph gland (LGF) were used as an in vitro model for lymphoid microenvironment and we tested their ability to inhibit spontaneous apoptosis of B-CLL cells. Co-culture of B-CLL cells with human BMFs, LGF, and SF significantly inhibited apoptosis and prolonged survival of the leukemic cells in comparison to suspension cultures and to co-cultures with fibroblasts obtained from non-lymphoid organs. Trans-well culture experiments indicated that cell-cell interaction and soluble mediators are essential for this supportive effect. To explore the involvement of PI3-K/Akt pathway in the anti-apoptotic effect of stromal fibroblasts, co-cultures were performed in presence of PI3-K inhibitors (wortmannin or Ly294002) or siRNAs against PI3-K (p110ß subunit) and Akt1. These inhibitors significantly reduced the supportive effect of stromal fibroblasts and induced apoptosis in B-CLL cells. Interestingly, the leukemic cells were far more sensitive to PI3-K inhibition than T cells, monocytes and fibroblasts. Induction of apoptosis was associated with a significant decrease in the intracellular PIP3, PI3-K, PDK1 and Akt1, NF-kappa B, IKK and de-phosphorylation/activation of tumor suppressor protein PTEN. Studies using phosphospecific anti-PTEN antibody demonstrated that PBMC of CLL patients (n=40) highly express a phosphorylated form of PTEN. The results demonstrate that the PI3-K/Akt pathway is involved in inhibition of apoptosis of B-CLL cells and suggest that interaction of the leukemic cells with lymphoid microenvironment maintains the activation of this pathway. The data also suggest that targeting this pathway represents a new option for designing novel therapeutic strategies in B-CLL.