bluuebunny/crossref_metadata_2025 huggingface dataset:
Unexpected Evidence That Dimethylsulphoxide (DMSO) Upregulates Expression of CXCR4 on Hematopoietic Stem/Progenitor Cells (HSPC), Increases Their Responsiveness to an SDF-1 Gradient and Enhances Homing to Bone Marrow.
Authors: Majka Marcin , Jarocha Danuta , Wysoczynski Marcin , Sag Duygu , Zuba-Surma Ewa , Kijowski Jacek , Gozdzik Jolanta , Ratajczak Mariusz Z.
Published: October 2019

Cryopreservation of bone marrow (BM), mobilized peripheral blood (mPB) and cord blood (CB) cells is a routine procedure to store hematopoietic stem/progenitor cells (HSPC) for transplantation. Dimethylsulphoxide (DMSO), the most commonly used cryoprotectant, is toxic to cells at higher concentrations (>10%); moreover, the freezing-thawing procedure itself is inevitably connected with the loss of HSPC. However, by chance we observed that short exposure of HSPC to DMSO enhances the responsiveness of these cells to an SDF-1 gradient and since SDF-1 is a major chemoattractant that navigates homing of HSPC to BM we became interested in elucidating this phenomenon. We found that short incubation (5–10 min) of human CB mononuclear cells (MNC) with DMSO at concentrations employed for cryopreservation (5–10%) significantly upregulates the expression of both CXCR4 (x 2–3) and CD34 (x 1.5) on CB MNC (as measured by FACS). Furthermore, DMSO significantly increased the chemotactic responsiveness (x 2–4) of CB MNC, BM MNC and selected CXCR4+ human hematopoietic cell lines (Jurkat, THP-1 cells) when the cells were exposed to 5–10% DMSO before chemotaxis assay. These responses to an SDF-1 gradient correlated with enhanced chemotaxis also of human CD34+, CD34+ CD38+, CD34+ CD38−, and CD34+ CXCR4+ clonogeneic progenitor cells, suggesting that DMSO directly enhances the responsiveness of human early progenitors (p<0.0001). At the molecular level, 5–10% DMSO strongly stimulated and prolonged SDF-1-dependent AKT phosphorylation. However, at the same time DMSO inhibited phosphorylation of MAPKp42/44. Similar observations were made for Sca-1+ BM-derived murine cells. In parallel experiments we found that murine Sca-1+ cells when preincubated with DMSO formed more 12 day-CFU-S colonies in spleens after transplantation into irradiated syngeneic recipients. Accordingly, x 2 more CFU-S were formed when Sca-1+ cells were exposed before transplantation to 5% DMSO and about x 4 more after exposure to 10% DMSO. Finally we employed a Ly5.1/Ly5.2 congeneic transplant model and showed that transplantation of Ly5.1 Sca-1+ cells exposed to 10% DMSO before transplantation resulted in higher chimerism in transplanted Ly5.2 mice as compared to untreated cells (control) (p<0.0001). In conclusion, we show for the first time an unexpected beneficial role of DMSO (5–10%) in regulation of homing of HSPC after transplantation and suggest that a short priming of HSPC with DMSO, even of non-cryopreserved cells, before transplantation may become a new strategy to enhance engraftment
Bcr-Abl Resistance Screening Predicts a Limited Spectrum of Point Mutations To Be Associated with Clinical Resistance to the Abl Kinase Inhibitor AMN107.
Authors: von Bubnoff Nikolas , Saenger Jana , Manley Paul W. , Mestan Juergen , Peschel Christian , Duyster Justus
Published: October 2019

In advanced-phase CML, resistance to imatinib mesylate is frequently associated with point mutations in the Bcr-Abl kinase domain. New, highly potent Abl kinase inhibitors such as AMN107 and BMS-354824, have recently entered clinical trials. Data from analyses of resistant patients will be available not before a large number of resistant patients will have been treated within clinical trials. Therefore, it will be important to generate specific resistance profiles for each compound prior to its therapeutic application. Using a cell-based screening method for resistance of Bcr-Abl positive leukemia to Abl kinase inhibitors, we generated a resistance profile for AMN107 and compared it to the resistance profile of imatinib mesylate. In contrast to imatinib, resistance to AMN107 was associated with a very limited spectrum of Bcr-Abl kinase mutations. While 26 exchanges at 21 positions occured with imatinib, the AMN107 screen revealed eight different exchanges at seven amino acid positions, with four exchanges affecting the P-loop. Novel mutations which have never been observed with imatinib, either in vitro or in resistant patients, emerged in the presence of AMN107 including an F359 exchange to isoleucine and a Q252H/S349L double mutant. In contrast to imatinib, the frequency of resistant colonies dramatically decreased with increasing AMN107 concentrations. Rarely emerging resistant colonies at a concentration of 400 nM AMN107 exclusively contained T315I. With the exception of T315I, all mutations that were identified were effectively suppressed when AMN107 was increased to 2000 nM, a concentration which is achieved in plasma in treated patients. Thus, in this system, increasing the AMN107 concentration to 400 nM prevented the emergence of resistant colonies, with the exception of T315I. Our findings suggest that AMN107 might be superior to imatinib in terms of the development of resistance. Also, AMN107 at clinically relevant concentrations may overcome imatinib resistant disease, including cases with expression of P-loop mutations. However, our study indicates that clinical resistance to AMN107 may be associated with the predominant emergence of T315I. Using this or similar approaches, it will be possible to provide information that translates into combinatorial and sequential treatment strategies and to determine critical plasma concentrations for mutations that might occur during treatment.
SPTBN1-FLT3 in Atypical Chronic Myeloid Leukemia Transforms Ba/F3 Cells to IL-3 Independence and Is Sensitive to Both Tyrosine Kinase Inhibitors and Immunotherapy.
Authors: Grand Francis H. , Iqbal Sameena , Zhang Lingyan , Russell Nigel H. , Chase Andrew , Cross Nicholas C.P.
Published: October 2019

We have identified a patient who presented with BCR-ABL negative chronic myeloid leukemia (CML) and an acquired 46XX, t(2;13;2;21) (p13;q12;q33;q11.2) in all bone marrow metaphases examined. Fluorescence in situ hybridization (FISH) using probes flanking the FLT3 gene at 13q12 suggested that this gene was disrupted. 5′-RACE PCR using primers to the region of FLT3 encoding the tyrosine kinase domain identified a novel in-frame mRNA fusion between exon 3 of SPTBN1 (spectrin, beta, non-erythrocytic 1 isoform 2, NM 178313) on chromosome 2p16 and exon 13 of FLT3 (NM 004119). Juxtaposition of SPTBN1 and FLT3 was confirmed by two color FISH and amplification of the genomic DNA breakpoint confirmed a fusion between intron 3 of SPTBN1 and intron 12 of FLT3. The SPTBN1-FLT3 fusion gene is predicted to be translated into a 570 amino acid chimeric protein that retains two coiled-coil domains from SPTBN1 and 424 amino acids from FLT3, including the entire tyrosine kinase domain. Since the t(2;13) is readily visible by cytogenetic analysis but has not been reported previously it seems likely that SPTBN1-FLT3 is uncommon. However to test if FLT3 might be involved more widely in BCR-ABL negative CML we analysed 40 cases by RT-PCR. Two cases were positive for the FLT3 internal tandem duplication (ITD) but mutation of residue D835 was not observed. Expression of the SPTBN1-FLT3 fusion transformed the interleukin 3 (IL-3)-dependent cell line Ba/F3 to growth factor independence and was accompanied by constitutive phosphorylation of the fusion protein and the downstream substrate ERK1/2. The growth of transformed cells was inhibited in a dose-dependent fashion by SU11567 and PKC142, but not by imatinib mesylate. The patient was initially treated with hydroxyurea and subsequently underwent an unrelated donor bone marrow transplant. She relapsed cytogenetically at 4 years but responded to donor lymphocyte infusion (DLI), achieving sustained cytogenetic and molecular (nested RT-PCR) remission. We conclude that SPTBN1-FLT3 is a rare abnormality in BCR-ABL negative CML that is responsive to both targeted signal transduction therapy and immunotherapy by DLI.
Early Bortezomib Following Allogeneic Stem Cell Transplantation (SCT) for Multiple Myeloma To Enhance or Maintain Remission Status.
Authors: Kroeger Nicolaus , Zabelina Tatjana , Ayuk Francis , Atanackovic Djordje , Schieder Heike , Renges Helmut , Zander Axel R.
Published: October 2019

We investigated the toxicity and efficacy of early administration of bortezomib in 14 patients with residual disease (n=9) or complete remission (immunofixation-negative) (n=5) after a median of 383 days (range, 47–1317) after melphalan/fludarabine-based dose-reduced allogeneic SCT for multiple myeloma. The median age was 49 years (range, 32–62), and stem cell graft was from HLA-identical siblings (n=2) or unrelated donors (n=12). Three patients had received prior Thalidomide and donor lymphocyte infusion, and 2 of them still received low dose thalidomide (50 mg) during bortezomib treatment. The study consisted of two cycles bortezomib (1.3 mg/m2) given intravenously on day 1, 4, 8 and 14 (q21d). In case of response and low toxicity, two further cycles were applied. Eleven patients completed two cycles, and 4 patients completed four cycles. Two patients stopped after three cycles due to side-effects; 3 patients could not complete the proposed two cycles and stopped either after the first injection (n=1) of the first cycle, or after the second (n=1) or third (n=1) injection of the second cycle due to neurotoxicity. Major toxicity was thrombocytopenia grade IV (> 75 % reduction) which was seen in 6 patients (43 %), but none required platelet support. Neutropenia grade IV was seen in 2 patients (14 %), neurotoxicity grade III and IV was observed in 3 patients (21 %) while mild grade I-II neuropathy was seen in 10 patients (71 %). Other toxicity was fatigue grade I (85 %), nausea grade I (50 %), diarrhea grade I-II (93 %). Two patients with acute or chronic GvHD of the skin experienced deterioration of the manifestation, which could be treated succesfully with corticosteroids. A significant decrease during bortezomib-therapy was observed for CD3+ cells (p=0.02), while no effect was seen for B-cells (CD19+) (p=0.4). Two patients who received bortezomib on day 125 and 201 after allogeneic SCT experienced Herpes zoster infection during and 2 weeks after bortezomib treatment, respectively. All 9 patients with persistent disease showed further regression of the paraprotein including negative immunofixation in 5 patients. We conclude that early bortezomib after allogeneic SCT for patients with multiple myeloma is active and can induce further regression of the paraprotein. The toxicity is considerable especially if given early after allo SCT.
High Frequency and Early Onset of Bone Mineral Density Loss Following Allogeneic Stem Cell Transplantation.
Authors: Dunford Lauren M. , Roy David M. , Hahn Theresa , Brown Karen , Paplham Pamela , Lamonica Dominick M. , Battiwalla Minoo , Padmanabhan Swaminathan , McCarthy Philip L.
Published: October 2019

Bone mineral density (BMD) loss has been recently associated with allogeneic stem cell transplantation (alloSCT). It is not certain whether BMD loss in alloSCT patients (pts) occurs primarily within the first six months, since some reports cite continuing BMD loss up to six years post-transplant. Furthermore, it has been suggested that alloSCT-BMD loss is more likely to manifest in the femoral neck (FN), as opposed to the lumbar spine (LS). There are no studies that have detailed the incidence of alloSCT-BMD loss. We retrospectively reviewed 90 alloSCT pts who underwent a single alloSCT between 7/1/2001 and 2/26/2005 to assess the frequency of DEXA scan use and alloSCT-BMD loss. 7/1/2001 was chosen as the start date since this was when DEXA scans were first available at RPCI. 50 patients did not have DEXA scans following BMT: 15/50 pts died before day 130, 21/50 pts died after day 130 post BMT, and 14/50 pts are alive post day 130 without DEXA scans (median 1.1 yrs, range 0.34–3.0 yrs). Clinical criteria for obtaining DEXA scans were a medically stable condition with a good KPS and alive at day 130. 37 pts met this criteria. The median survival of the non-DEXA scan pts was 180 (7–1296) days and 571 (110–1282) days for the DEXA scan pts. DEXA scan pt characteristics in this sample included: Median age 39 years (range 7–66); AML (n=12), Lymphoma (n=12), CML (n=5), MDS/MPD (n=5), ALL (n=2), or Aplastic Anemia (n=1); Flu+Mel (n=14), Bu+Cy (n=8), Cy+TBI (n=7), non-myeloablative regimen (n=6), or other regimens (n=2); FK+Mt (n=28), FK+/− other (n=4), Mt (n=2), Cs+Mt (n=2), or Cs+FK+Mt (n=1). Approximately half (n=18/37) of alloSCT pts were found to have BMD loss at day 130 post-transplant. Results were stratified by year, and no trends regarding the frequency of DEXA scans in the population or the frequency of abnormal results over time were noted. There were no differences in terms of conditioning regimens or GVHD prophylaxis between the normal and low BMD groups. Contrary to previously published reports, we did not find a marked difference between the LS and FN T scores of the 18 pts with BMD loss at baseline (LS median T score = −1.4, FN median T score = −1.55). Also, BMD improvement following oral bisphosphonates was seen in six pts with available follow-up scans (LS median T score improved from −1.15 to −0.55, FN median T score improved from −1.45 to −1.1). The day 130 incidence of BMD loss in our alloSCT population was higher than we expected. Therefore, we recommend that all future alloSCT pts undergo DEXA scans as part of the day 100 workup. Further study may elucidate risk factors for BDM loss post BMT. Bisphosphonate use may reverse alloSCT BDM loss.
PI3K/Akt/FOXO3a Pathway Contributes to Thrombopoietin-Induced Proliferation of Primary Megakaryocytes In Vitro and In Vivo Via Modulation of p27Kip1.
Authors: Nakao Takafumi , Geddis Amy E , Fox Norma E. , Kaushansky Kenneth
Published: October 2019

Thrombopoietin (TPO), the primary regulator of megakaryocyte (MK) and platelet formation, modulates the activity of multiple signal transduction molecules, including those in the Jak/STAT, p42/p44 MAPK, and phosphatidylinositol 3-kinase (PI3K)/Akt pathways. In the previous study, we reported that PI3K and Akt are necessary for TPO-induced cell cycle progression of primary MK progenitors. The absence of PI3K activity results in a block of transition from G1 to S phase in these cells (Geddis AE et al. JBC2001276:34473–34479). However, the molecular events secondary to the activation of PI3K/Akt responsible for MK proliferation remain unclear. In this study we show that FOXO3a and its downstream target p27Kip1 play an important role in TPO-induced proliferation of MK progenitors. TPO induces phosphorylation of Akt and FOXO3a in both UT-7/TPO, a megakaryocytic cell line, and primary murine MKs in a PI3K dependent fashion. Cell cycle progression of UT-7/TPO cells is blocked in G1 phase by inhibition of PI3K. We found that TPO down-modulates p27Kip1 expression at both the mRNA and protein levels in UT-7/TPO cells and primary MKs in a PI3K dependent fashion. UT-7/TPO stably expressing constitutively active Akt or a dominant-negative form of FOXO3a failed to induce p27Kip1 expression after TPO withdrawal. Induced expression of an active form of FOXO3a resulted in increased p27Kip1 expression in this cell line. In an attempt to assess whether FOXO3a has an effect of MK proliferation in vivo, we compared the number of MKs in Foxo3a-deficient mice and in wild type controls. Although peripheral blood cell counts of erythrocytes, neutrophils, monocytes and platelets were normal in the Foxo3a-deficient mice, total nucleated marrow cell count of Foxo3a-deficient mice were 60% increased compared with wild type controls. In addition, the increase of MKs was more profound than that of total nucleated marrow cells; CD41+ MKs from Foxo3a-deficient mice increased 2.1-fold, and mature MKs with 8N and greater ploidy increased 2.5-fold, compared with wild type controls. Taken together with the previous observation that p27Kip1-deficient mice also display increased numbers of MK progenitors, our findings strongly suggest that the effect of TPO on MK proliferation is mediated by PI3K/Akt-induced FOXO3a inactivation and subsequent p27Kip1 down-regulation in vitro and in vivo.
Long-Term Outcome after Bone Marrow Transplantation for Adult Thalassemia.
Authors: Gaziev Javid , Sodani Pietro , Roveda Andrea , Spitaleri Luca , Montuoro Aldo , Simone Maria Domenica , Erer Buket , Polchi Paola , Lucarelli Guido
Published: October 2019

Advances in management of thalassemia have increased the proportion of patients living into adulthood. However, disease and treatment related complications in these patients progress over time causing severe morbidity and shortened life expectancy even in well-resourced countries with universal access to good medical treatment. Adult thalassemics constitute a high risk group of patients for transplantation due to both advanced age and acquired multiple organ damage. We present here long-term outcome after BMT for adult thalassemia and a new approach to transplant adult patients. Between November 1988 and September 1996, 107 consecutive patients (median age 22 years; range 17–35) received BMT from HLA-identical related donors following BU14/16 CY120/160 regimen. There were 18 class 2 and 89 class 3 patients. The probability of overall survival, event-free survival, mortality and rejection were 66%, 62%, 37% and 4% respectively. Interestingly, unlike class 3 younger patients (age <17 years) adult patients treated with the same treatment regimens had a low probability of rejection suggesting that donor-host tolerance might be induced even among extensively transfused patients. The incidence of grade 2–4 acute GVHD was 24.5%, and moderate or severe chronic GVHD was 5.6%. The presence of chronic active hepatitis at the time of transplant was the only factor associated with poor survival (RR 2.5; p=0.05). Sixty eight patients survive with a median follow-up of 12 years (range 8.3–16.2), and 66 of them are free of thalassemia. Current myeloablative BMT in adult patients is characterised by higher transplant related mortality due to the advanced stage of disease. Therefore, new approach to SCT that might reduce transplant related morbidity and mortality in adults is warranted. In 1997 we devised a new treatment protocol (Protocol 26) for adult thalassemics aimed at reducing the bone marrow and gradually increasing immunosuppression before transplant to avoid peritransplant drug toxicity. Patients were given hydroxiurea 30 mg/kg/day, azathioprine 3 mg/kg/day from day -45 through day -11, and fludarabine 20 mg/m2/day from day -17 through day -13 along with hypertransfusion and continuous chelation. On day -9 they started a reduced dose intensity conditioning regimen with BU14 CY90. Fifteen higher risk patients (median age 21 years; range 17–31) with class 3 thalassemia (n=14) or sickle cell anemia (n=1) were given BMT from HLA-identical siblings after preparation with this new protocol. Overall, treatment was well tolerated with no increase in toxicity. The probability of thalassemia-free survival and transplant related mortality were 67% and 27% respectively. Our new approach to transplant adult patients with a reduced dose intensity conditioning regimen showed some improvement in thalassemia-free survival but transplant related mortality in these high risk patients still remains elevated.
Hematocrit and Oxygen Dependence of Nitric Oxide Scavenging by Red Blood Cells.
Authors: Kim-Shapiro Daniel B. , Azarov Ivan , Huang Kris T. , Hogg Neil , Gladwin Mark T.
Published: October 2019

Human, animal and in vitro studies suggest that reactions involving nitric oxide (NO) and cell-free hemoglobin (Hb) occur up to 1000 times faster than those with red blood cell (RBC) encapsulated hemoglobin. Thus, cell-free Hb resulting from hemolysis or infusion upsets vascular homeostasis and contributes to pathological conditions in hemolytic anemias such as sickle cell disease and paroxysmal nocturnal hemoglobinuria. The slow NO uptake by RBCs results either from internal factors such as a physical barrier set up by the RBC membrane or external factors such as the time required for NO to diffuse to the cell. We have conducted experiments in which both external and internal factors play a role in limiting NO uptake wherein excess NO is rapidly mixed with RBCs and NO uptake is monitored by absorption spectroscopy. In addition, we have measured NO uptake under conditions where the role of the external factors are reduced by having cell-free and RBC encapsulated Hb compete for limited amounts of slowly released NO, measuring the reaction products using electron paramagnetic resonance spectroscopy. We are the first to have conducted these types of measurements at physiologically relevant hematocrits. Our major findings are that (1) the relative rate that deoxygenated red blood cells scavenge nitric oxide is 3–8 times faster than oxygenated red cells; surprisingly, this effect appears to be partially attributable to a physical membrane barrier, and (2) the rate of NO scavenging by oxygenated RBCs increases significantly (about 3 fold) when the hematocrit is raised from 15% to 50%. Our biophysical data support the thesis that cell-free Hb is capable of greatly reducing the bioavailability of NO and thereby negatively effect hemodynamics and endothelial function. The hematocrit dependence of NO scavenging by intact erythrocytes provides a potential pathogenic mechanism for hypertension during erythropoietin therapy, mortality associated with eNOS inhibition in polycythemic mice, and endothelial dysfunction in acquired polycythemic conditions. The observed oxygen dependence of NO uptake rate supports models in which the submembrane red cell protein scaffolding contributes to NO homeostasis, but not to the extent that has been previously proposed by others. Modulation of the effects of the scaffolding on scavenging and export of NO activity by RBCs under normoxic and hypoxic conditions requires further investigation.
Is Antilymphocyte Globulin Useful in Conditioning Regimen of Unrelated Donor Hematopoïetic Stem Cell Transplantation?.
Authors: Esperou Helene , Appert Marie Lorraine , Agha Ibrahim Yacoub , Milpied Noel , Ifrah Norbert , Kuentz Mathieu , Chir Zina , Raffoux Colette
Published: October 2019

Between 1998 and 2004, 2141 allogeneic hematopoietic stem cell transplantations with unrelated donor (excluding cord blood) have been recorded on the SFGM-TC data base. The lack of unique behavior in regard of Anti Lymphocyte Globulin (ALG) use in unrelated donor transplants allowed us to perform a retrospective study to assess the influence of ALG in such grafts. To avoid biaises we excluded T cell depletion or monoclonal antibody use, reduced intensity conditioning and focused only on patients (pts) with acute leukemia (AL) and myelodysplasia (MDS). So we defined a study population of 553 pts. In addition, in order to analyze the actual impact of ALG on wellknown targets (engraftment, acute GvHD, relapse, survival) we choose to keep only the cases in which was available the allelic HLA typing (4 digits) for the recipient and the donor, i.e. 250 pts. There were 106 females and 144 males, allografted in 29 centers. The median age was 23 years (1–62) and there were 77 children under 15. There were 223 AL - myeloid n=120, lymphoid n=103 - and 27 MDS. The source of cells was bone marrow for 190 pts and peripheral mobilized stem cells for 60. One hundred and ninety-three pts received a conditioning regimen including Total Body Irradiation. Ninety-five pts received ALG (mainly Thymoglobulin Mérieux-Genzyme Lyon France at various doses) for a median of 3 days (group 1) and 155 did not (group 2). There were 189 donor/recipient pairs strictly HLA-matched 10/10, 49 with one allelic difference (HLA A n=13; HLA B n=8; HLA C n=13; HLA DRB1 n= 5; HLA DQB1 n=10 ), 11 with 2 allelic differences and 1 with 3 allelic differences. 238 pts have sustained engraftment at a median time of 20 days (9–41) without any influence of ALG use (97% vs 94% NS). Overall survival at 3 years was 45% in group 1 and 52% in group 2 (NS). The incidence of acute GvHD (grade II to IV) was similar in the two groups (59% vs 54%), but the incidence of acute GvHD (grade III to IV) was lower in group 1 (21% vs 32% p=0.03). Use of ALG did not affect rate relapse (18% vs 20% NS). In a multivariable analysis including HLA disparities, patient age, use of ALG, two factors appear as predictive for grade III–IV GvHD: HLA-identity (p=0.05) and use of ALG (p=0.02). These factors are not significant for survival in the same multivariable analysis but in this allelic matched population, the influence of ALG should be analyzed in regard of exact dose of ALG.
Final Report on Tandem Autologous Stem Cell Transplantation for Patients with Primary Progressive or Poor Risk Recurrent Hodgkin Lymphoma - A Two Institution Study.
Authors: Fung Henry C. , Stiff Patrick , Nademanee A. , Smith E. , Cutrone C. , Parthasarathy M. , Krishnan A. , Klein J. , Molina A. , Ruel C. , Smith D. , Rodriquez T. , Falk P. , Toor A. , Schriber J. , Rosenthal J. , Ivers B. , Somlo G. , Kogut N. , Vora N. , Margolin K. , Spielberger R. , Forman Stephen J.
Published: October 2019

Background: While autologous stem cell transplantation (ASCT) for patients with relapsed/refractory Hodgkin lymphoma (HL) appears to offer a survival advantage over conventional therapy, only approximately 25–35% of patients with primary progressive or poor risk recurrent Hodgkin Lymphoma achieve durable remission after ASCT with disease progression after transplant accounted for most of the treatment failures. We (a collaboration between City of Hope Comprehensive Cancer Center and Loyola University Medical Center) conducted a pilot study to evaluate the toxicities and efficacy of a tandem transplant approach in this subgroup of patients.

Methods: Between 4/98 and 3/00, 46 patients (median age: 37 [range 17–65]) were enrolled in the study.

Eligibility criteria: primary progressive (n=28) or recurrent HL (n=18) with at least one of the following poor prognostic factors: first CR < 12 months (n=15) or extra-nodal disease (n=4) or B symptoms at relapse (n=4). The 1st cycle consisted of melphalan (150mg/m2) alone. The 2nd cycle consisted of Fractionated total body irradiation (1200cGy) or BCNU (450mg/m2) in combination with etoposide (60mg/kg) and cytoxan (100mg/kg). A minimum of 2 x 106 CD 34/kg autologous stem cells were re-infused after each cycle of therapy.

Results: Of the 46 pts, 41 (89%) patients completed the planned tandem transplants. Five (11%) did not receive the planned tandem transplants because of inadequate stem cells collection for 2nd ASCT. After a median of 64 days (25–105), 41 patients received the 2 transplants. With a median follow up of 5 years (1.6–6.4), 24 patients are alive and progression-free at last follow up. Of the 41 patients who received both cycles of high dose therapy, 21 are alive and disease-free; 17 developed disease progression at a median of 4 months (range 1–61) post-ASCT. There were 2 early deaths secondary to regimen related toxicities. Both of them died of interstitial pneumonitis. The Day100 mortality was 4%. One patient developed secondary MDS 28 months post-ASCT after receiving further chemotherapy for relapsed disease. Using an intent-to-treat analysis for all 46 patients included in the study, the 5-year K-M estimate of overall survival, progression free survival and event free survival were 73% (95%CI 60,87), 61% (95% CI 46, 76) and 50% (95% CI 35, 64) respectively.

Conclusion: Our mature results from this study suggest that in patients with primary progressive or poor risk recurrent Hodgkin Lymphoma, this tandem ASCT program is effective and well tolerated and compares favorably with the conventional single transplant without increase in transplant related toxicities.
Prolonged Survival in Patients with Angioimmunoblastic T-Cell Lymphoma (AIL) after High-Dose Chemotherapy and Autologous Stem Cell Transplantation (ASCT). The GELTAMO Experience.
Authors: Rodriguez Jose , Caballero Maria Dolores , Gutierrez Antonio , Gandarillas Marco , Leon Angel , Ojanguren Jesus , Sureda Ana , Lopez-Guillermo Armando , Carrera Dolores , Bendandi Mauricio , Arranz Reyes , Moraleda Jose , Ribera Jose Maria , Albo Carmen , Morales Alfonso , Garcia Juan Carlos , Fernandez Pascual , Cañigral Guillermo , Bergua Juan , Conde Eulogio
Published: October 2019

Conventional treatment of patients with AIL offers poor results with a median survival ranging from 15 to 24 months. No standard treatment for this disease has been established. Thus, our aim in this report is to provide our experience within a cooperative group in patients with this type of lymphoma who underwent ASCT. Nineteen patients were transplanted in the GELTAMO cooperative group in the last 12 years. Median age at transplant was 46 years. Fifteen patients (79%) underwent the procedure as front line therapy, 8 out of these 15 pts as consolidation of first CR, 5 in first PR and 2 in induction failure condition. Four patients who relapse received the procedure as salvage therapy. Most patients (90%) received peripheral stem cell product and the conditioning regimen was based in BEAM or BEAC in 79% of patients. Seventy-nine percent of patients achieved a CR with the transplant, 5% a PR and 16% of patients failed the transplant. With a median follow up of 25 months for alive patients, 11 patients are alive and free of disease at present. The OS at 5 years is 60% and the PFS, 55%. Eight patients died, 7 of them of disease and 1 patient of a second neoplasia. Prognostic factors associated with a poor outcome were the presence of extranodal disease at transplant, bone marrow involvement, the presence of more than 1 factor of the age-adjusted-IPI (a-IPI) and be transplanted in refractory disease. Patients with AIL who underwent ASCT in chemosensitive disease have 60% of probability to be alive at 5 years, most of them free of disease. Patients who received the procedure as consolidation either as first CR or in first PR have similar outcome. However patients in refractory disease do not benefit of this therapeutic modality. The presence of extranodal disease at transplant predicts an unfavourable outcome.
Prognostic Factors in the UK LRF CLL4 Trial.
Authors: Oscier David Graham , Richards Sue , Orchard Jenny , Davis Zadie , Best Giles , Morilla Alison , Brito-Bapapulle Vasantha , Matutes Estella , Pettitt Andrew , Catovsky Daniel
Published: October 2019

The CLL4 trial randomised 777 previously untreated patients between Chlorambucil (Chlor), Fludarabine (Fluda) and Fludarabine with Cyclophosphamide (FC). The median follow-up is 21 months. VH gene mutation status using a 98% cut off, CD38 expression using a 7% cut off, ZAP70 expression (Orchard et al Lancet 2004) and interphase FISH were measured on samples taken at randomisation. In an interim analysis 253/397 patients had unmutated VH genes. 32 patients utilised the VH3-21 gene, of whom 27 showed restricted HCDR3 usage. 343/535 were CD38 positive and 131/261 were ZAP70 positive. 10/24 VH 3-21 cases were ZAP 70 positive, of whom 7 were unmutated. There was no significant association between VH genes and age, sex or a positive DAT. There were significant correlations between VH gene mutations and CD38, ZAP70 and FISH results.

Association of VH genes with CD38, ZAP70 and FISH results. CD38+ ZAP 70+ p53 loss Del 11q Del 6q21 Del 13q Trisomy 12 % = % of all cases with known VH mutation status VH Unmutated 119 (58%) 110 (72%) 21 (9%) 48 (22%) 18 (10%) 109 (45%) 50 (21%) VH Mutated 30 (25%) 17 (16%) 3 (2%) 13 (11%) 6 (6%) 97 (73%) 13 (10%) p= <0.0001 <0.0001 0.02 <0.009 0.2 <0.0001 0.008

Logistic regression including age, stage, gender, VH mutations and genetic factors shows that stage (p=0.002), VH genes (p=0.05), p53 loss (p=0.02) and del 11q (p=0.01) affect response. Response rates by VH gene mutation status and treatment arm are given in table 2.

Response rates according to VH mutation status and treatment arm Chlor Unmut Chlor Mut Fluda Unmut Fluda Mut F/C Unmut F/C Mut %=% of all unmutated or mutated cases receiving a particular treatment CR/NPR 24 (25%) 23 (42%) 25 (43%) 18 (55%) 37 (58%) 23 (70%) PR 43 (45%) 19 (34%) 21 (36%) 10 (30%) 21 (33%) 10 (30%) NR/PD 29 (30%) 13 (24%) 12 (21%) 5 (15%) 6 (9%) 0 (0%) p (trend) 0.07 0.3 0.1

Analysis of variance shows that both VH genes (p=0.01) and treatment (p<0.0001) have an effect on response with no significant interaction. Among 104 patients with unmutated VH genes and both ZAP 70 expression and response data available, there was no significant difference in response rates between ZAP positive and negative cases. In univariate analysis p53 loss (p<0.00005), unmutated VH genes (p=0.003), CD38 positivity (p=0.06) but not ZAP70 positivity correlate with poor overall survival. In multivariate Cox regression analyses, age (p<0.0001) stage (p=0.01) and p53 loss (p<0.0001) are significant for survival. When patients with p53 loss are excluded unmutated VH genes (p=0.007) but not CD38 or ZAP70 positivity are significant. In conclusion p53 loss identifies a small group of patients with both poor response to treatment and short survival. In the remaining cases, unmutated VH genes are associated with a relatively poor response independent of treatment arm, and with a shorter survival.
Transcriptosome Profiling of B-CLL Identifies WNT-3A and ROR-1 as an Autocrine Mechanism in Cell Survival.
Authors: Choi James , Simons B. , Riley Chris , Klinkhammer T. , Cooke Laurence , Spier Catherine , Sud R. , Hehn Sean , Garewal Harinder , Mahadevan Daruka
Published: October 2019

Background: B-cell chronic lymphocytic leukemia (B-CLL) is the most common leukemia afflicting the Western world. B-CLL accounts for 25% of all newly diagnosed leukemias. Despite many new therapeutic advances, B-CLL is still not a curable malignancy. The hallmark feature is the presence of an elevated number of circulating clonal leukemic B cells that typically express CD 5, CD 19, CD 23, and low levels of surface immunoglobulins.

Methods: Mononuclear cells from 5 patients were analyzed utilizing the HG-U133A 2.0 Affymetrix array (~18,400 transcripts, 22,000 probe sets) after isolating and purifying total RNA (Qiagen, RNAeasy). The control RNA samples were isolated from normal peripheral blood (PB) B-cell (AllCell, CA). Immunohistochemistry (IHC) confirmed tumor lineage and quantitative real time RT-PCR was performed on selected genes to validate the microarray study. The GEP data was processed and analyzed utilizing Affymetrix MAS 5.0 and GeneSpring 5.0 software. Our data was analyzed in the light of published GEP of B-cell CLL. Fifteen B-CLL patients (retrospectively) were evaluated by RT-PCR for ROR-1 and WNT-3A with gene specific probes. As a potential therapy, thalidomide was evaluated on B-CLL cells grown in cell culture for 24 hours. GEP of the thalidomide treated B-CLL from the initial 5 patients was performed to look for gene expression changes that could drive the B-CLL toward apoptosis. A homology model of ROR-1 tyrosine kinase was built, ATP docked and in silico databases screened for potential lead molecules.

Results: Data are represented as “robust” increases or decreases of relative gene expression common in the 5 patients. However, ROR-1 and WNT-3A were consistently over-expressed together in these 5 patients. The average increase was 25-fold for ROR-1 and 7-fold by WNT-3A when compared to normal B-cell RNA. Of the 15 patients we evaluated for ROR-1 and WNT-3A with gene specific probes, the increase in gene expression correlated well with our initial gene expression profiling study. Thalidomide specific gene changes included several molecules involved in apoptosis. Of these gene changes, Bcl-G, p35, and Cdk-5 were up-regulated several fold. Data will be presented on the influence of the stage of disease on ROR-1 and WNT-3A expression.

Conclusions: GEP of B-CLL in combination with quantitative real time RT-PCR has identified several novel therapeutic targets for therapy based on a comparison to normal (B-cell) RNA. GEP has identified ROR-1 as a key component in an autocrine pathway that helps B-CLL elude apoptosis. The identification of this novel tyrosine kinase-like protein has led to the development of a molecular target for future therapeutic applications. Several lead compounds have been identified and are being evaluated as potential therapeutics in B-CLL.
Development of Forodesine Hydrochloride (FH), an Inhibitor of Purine Nucleoside Phosphorylase, for Patients with Chronic Lymphocytic Leukemia (CLL).
Authors: Balakrishnan Kumudha , Ravandi Farhad , Keating Michael J. , Gandhi Varsha
Published: October 2019

Mammalian purine nucleoside phosphorylase (PNP) catalyzes the cleavage of inosine, deoxyinosine, guanosine, and deoxyguanosine (dGuo) to their corresponding base and sugar 1-phosphate by phosphorolysis. PNP deficiency in humans produces a relatively selective depletion of T cells without much effect on normal B cells. FH, a potent inhibitor of PNP, was designed based on the transition-state analog structure stabilized by the enzyme. Previous studies demonstrated that FH in the presence of dGuo inhibits the proliferation of T-lymphocytes (Kicska et al. PNAS 2001). Based on these observations, we conducted a phase I clinical trial of FH in patients with advanced T-cell malignancies. Significant antileukemic activity was correlated with an increase in plasma FH (median 5 μM) and dGuo (median 14 μM), and an accumulation of intracellular dGuo triphosphate (dGTP) (Gandhi et al, Blood, in press, 2005). High accumulation of dGTP in T-cells may be dependant on activity of deoxynucleoside kinases. Because B-CLL cells have high activity of deoxycytidine kinase, we hypothesized that they would be sensitive to FH. This postulate was tested in primary CLL lymphocytes during in vitro investigations. Lymphocytes from patients with CLL were incubated in vitro with FH (2 μM) in the presence of 10 μM dGuo. Lymphocytes from 3 of 4 patients showed an elevation in the intracellular dGTP levels to a median 30-fold at 8 hr, without any effect on other deoxynucleotides. This increase in dGTP was associated with phosphorylation of p53 at ser15, stabilization of p53, and an increase in p21 protein. The dGTP accumulation was related to induction of apoptosis measured by activation of caspase 8, 9, and 3 and cleavage of PARP. Incubation with either FH or dGuo alone did not result in dGTP accumulation or cell death suggesting that PNP inhibition by FH and phosphorylation of dGuo to dGTP are essential for CLL cell death. Based on these encouraging results, availability of oral formulation and to validate these in vitro data during clinical trial, a phase II study of FH in patients with advanced, fludarabine-refractory CLL has been initiated. FH is administered orally at a dose of 200 mg/day for 7 days each week for 4 weeks (cycle 1). Patients are evaluated after 1 full cycle of therapy and in the absence of serious side effects, or disease progression, they continue the treatment for up to 5 more cycles. Laboratory endpoints such as level of FH and dGuo in plasma, PNP activity, dGTP levels in cells, will be measured and correlated with cytoreduction. It is postulated that a high kinase/low nucleotidase activity leading to the accumulation of intracellular dGTP in target CLL cells and apoptosis (akin to what has been seen in T-cells) will result in response to therapy. This is the first trial of a PNP inhibitor for treatment of B-CLL.
Polymorphisms in the Promoter Region of the IL-10 Gene Is Associated with Inhibitor Development in Hemophilia A.
Authors: Astermark Jan , Oldenburg Johannes , Pavlova Anna , Berntorp Erik , Lefvert Ann-Kari ,   MIBS Study Group
Published: October 2019

The development of inhibitory antibodies is a severe and costly complication to replacement therapy occurring in 10–15% of patients with hemophilia A, and the aim of the Malmo International Brother Study (MIBS) is to evaluate host genetic factors associated with this adverse effect of treatment. In the present study, factor VIII mutations, HLA genotypes and polymorphisms of the interleukin IL-1beta, IL-4 and IL-10 genes known to influence antibody production in autoimmune diseases, were analyzed in 164 patients with hemophilia A (120 severe, 30 moderate and 14 mild) belonging to 78 unrelated families. Seventy-seven (47.0%) of the subjects had a history of inhibitors (57 high-responding, 20 low-responding) in 54 unrelated families (34 discordant, 20 concordant siblings). In 24 families, no inhibitor was reported in any of the siblings. Seventy-five patients (45.7%) in 36 families had an inversion. In this group, 40 patients (53.3%) in 28 families had inhibitors (17 concordant, 11 discordant). Weak associations between inhibitor development and the HLA alleles A26 and B44 were found. No association was found with the IL-1beta Taq 1 RFLP alleles in exon 5, and the −590 C/T SNP in the promoter region of IL-4. There was however, a strong association between an allele with 134 bp in one of the CA repeat microsatellites, IL-10G, located in the promoter region of the IL-10 gene, and development of inhibitor. Allele 134 was found in 32 (41.6%) of the patients with inhibitors compared with 12 (13.8%) of the inhibitor negative patients (p<0.001), corresponding to an odds ratio of 4.4 (95% CI 2.1–9.5, p<0.001). The association was consistent in the subgroup of families with severe hemophilia and an inversion of the factor VIII gene (p=0.002). Only one discordant inhibitor family was identified in which the subject without allele 134 developed an inhibitor, and the allele 134 positive brother did not. IL-10 is the first gene located outside the causative factor VIII gene mutation to be associated with inhibitor development in hemophilia and our data indicate this gene to be an important determinant for this side-effect of replacement therapy.
Fludarabine Versus Fludarabine Plus Epirubicin in the Treatment of Chronic Lymphocytic Leukemia - Final Results of a German Randomized Phase-III Study.
Authors: Rummel Mathias J. , Stilgenbauer Stephan , Gamm Heinold , Rost Andreas , Hartmut Doehner , Hoelzer Dieter , Bergmann Lothar
Published: October 2019

Based on promising results from our previous phase-II study demonstrating an overall response rate (ORR) of 92% and a high rate of complete remissions (CR) of 40% we initiated a multicenter randomized phase-III study to compare efficacy and toxicity of the combination fludarabine plus epirubicin (FluEpi) versus monotherapy with fludarabine (Flu) as first-line therapy or therapy of first relapse. In addition, cytogenetic aberrations were investigated in all patients to evaluate, if special chromosomal abnormalities are associated with prognosis and response to fludarabine. Treatment schedule: Fludarabine 25 mg/qm day 1–5, epirubicin 25 mg/qm day 4+5 for a maximum of 6 cycles every four weeks. Results: 150 patients (pts) are evaluable for response (77 pts with Flu, 73 pts with FluEpi). Median age of the patients is 62 years. All pts were in need of treatment with 86 pts being in stage Binet B and 64 pts in Binet C. Cytogenetic aberrations were well balanced between both groups. The ORR for the total group of 150 pts was 79% with a CR-rate of 19%. The ORR for patients treated with FluEpi was 88% compared to patients treated with Flu monotherapy reaching an ORR of 73%. This difference is statistical different with a p-value of 0.0255. CR are different in favor for combined treatment modality (29% CR for FluEpi vs 9% for Flu) with a significance level of 0.0029. Patients treated with FluEpi have a longer median duration of progression free survival with 26 months in comparison to 20 months for patients treated with fludarabine alone (p=0.085). There is a trend for a longer median overall survival time, which is 76 months for patients with FluEpi compared to the median survival time of 63 months for the Flu monotherapy group, however this difference is not statistical different (p=0.10). The event free survival (EFS), which was defined as not achieving any remission, progression/relpase of disease or death for any cause, was superior for pts treated with FluEpi (30 months) in comparison to the pts treated with Flu monotherapy (19 months) with a significance level of 0.048. Conclusions: The combination therapy of fludarabine plus epirubicin achieves statistically higher response rates and a longer duration of event free survival, however this does not translate in an statistical significant overall survival benefit.
Phase 2 Study of Talabostat and Rituximab in Patients with Advanced Chronic Lymphoctyic Leukemia (CLL) Previously Treated with a Rituximab/Fludarabine Regimen.
Authors: Khan Khuda D. , O’Brien Susan , Rai Kanti R. , Brown Jennifer R. , Cook Perry , Maddox Anne Marie , Duarte Alison M. , Haltom Eric J. , Uprichard Margaret J.
Published: October 2019

Background: Talabostat (T) is an orally available inhibitor of dipeptidyl peptidases (DPP) such as fibroblast activation protein (FAP). FAP is expressed in bone marrow, lymph nodes, and the stroma of solid tumors. T induces the production of cytokines and chemokines in lymph nodes and spleen, stimulating both adaptive and innate immune responses. T was shown to enhance the activity of rituximab (RTX) in a Phase 1 study in previously treated patients with B-cell malignancies, most likely by enhancing the antibody-dependent cytotoxicity of RTX. This study was conducted to evaluate the activity of T and RTX in patients with CLL who had previously failed a fludarabine/RTX (FR)-based regimen.

Methods: This is a single-arm, open-label study in up to 54 patients using the Simon 2-stage design. The 28-day treatment course consists of RTX 375mg/m2 on Days 1, 8, 15, and 22, with T 300mcg BID tablets for 6 days following each RTX infusion; additional courses are permitted at the discretion of the investigator depending on response. Eligibility criteria include: CD20(+) B-CLL; Rai Stage III or IV, or Stages I and II with lymphadenopathy or hepatosplenomegaly; ECOG 0-2; primary resistance or PD following a FR-regimen; no CNS metastases. The primary endpoint is disease response (evaluated per NCI-WG criteria); secondary endpoints include response duration, progression-free survival, and survival.

Results: Of 20 patients treated to date, 19 patients (17 men, 2 women; median age 62, range 44 to 80) have completed at least 1 course of treatment and are considered evaluable for response:17 patients failed prior FR and 2 patients failed prior F (RTX naive). A partial response (PR) is reported in 4/19 patients (21%); 3 who had failed FR and one who had falied F. In 3 patients, the PR was reported at Day 29. One patient who failed FR was noted to be a PR after multiple courses. At Day 29, stable disease was reported in 12 patients and 4 had progressive disease. Most patients have received 1 course of treatment; 2 have received 2 courses and one PR has received 5 courses. The most frequent adverse events (AEs) include: edema (47%), pyrexia (35%), fatigue (24%), and dyspnea (24%). Grade 3 dyspnea has been reported in 2 patients. Grade 4 events include decreased platelets in one patient and hypoglycemia in one patient with a PR who was taking antidiabetic medications. Hypoglycemia is consistent with the inhibition of DPP-IV by T.

Conclusion: The addition of T to RTX shows promising activity in advanced CLL, with clinical responses reported in patients who failed an FR-based regimen. Edema was the most frequent AE, and other AEs are consistent with the safety profile of RTX. Updated results will be presented at the Annual Meeting.