bluuebunny/crossref_metadata_2025 huggingface dataset:
Identification of Off-Patent Drugs That Reverse Daunorubicin Efflux Mediated by ABCB1 in T-ALL Cells.
Authors: Winter Stuart S. , Lovato Debbie M. , Khawaja Hadya M. , Edwards Bruce S. , Steele Irena D. , Young Susan M. , Oprea Tudor I. , Sklar Larry A. , Larson Richard S.
Published: October 2019

In acute leukemias, the overexpression of P-glycoprotein, encoded by the ATP Binding Cassette B1 (ABCB1) gene contributes to multidrug resistance (MDR), and is considered one of the major obstacles to successful cancer chemotherapy. The identification of small molecules that reverse ABCB1 efflux activity is critical for the successful treatment of cancers, including relapsed T-lineage acute lymphoblastic leukemia (T-ALL). However, the dose-limiting toxicities of many MDR reversal agents have restricted their use in clinical trials, and more effective and clinically applicable reversal agents remain to be identified. We have recently developed a T-ALL cell line that overexpresses ABCB1 and exhibits multiple drug resistance (MDR) to daunorubicin, prednisolone, and vincristine, but not L-asparaginase. The MDR can be reversed by suppression of ABCB1 expresssion with siRNA or 5 μM cyclosporine (CSA). Using this cell line, we developed a flow cytometry based, high-throughput screening assay that quantifies ABCB1 efflux using the fluorescent probe JC-1 (Swerts, et al Leuk Lymphoma 45:2221–8, 2004). We screened a library of 880 off-patent drugs for their ability to inhibit ABCB1 efflux at concentrations of 4 μM, and 19 compounds were identified, including CSA. Based on a published record of safe internal use in humans, 12 compounds were retained for further analysis (3 compounds were originally described as calcium channel blockers, 1 as sodium channel blocker, 1 as ACE inhibitor, 1 as dopamine uptake inhibitor, 1 as Topoisomerase II inhibitor, 2 as steroids, 2 as antifungal and 1 as immunosuppressant). We determined the 50% inhibitory concentration (IC50) of drug for ABCB1 efflux, the efflux reversal concentration of drug that rescued DNR-induced T-ALL cell death (ECrev50), and the corresponding in vitro toxic dose in 50% of treated T-ALL cells (TD50). Due to space limitations, detailed data could not be presented in the abstract. To our knowledge, 8 of the 12 compounds have not been previously described as ABCB1 inhibitors. These compounds may be useful as chemosensitizers or as lead compounds for development of improved ABCB1 inhibitors in T-ALL as well as other cancers.
Decitabine Induces High Response Rates in Patients with Chronic Myelomonocytic Leukemia (CMML).
Authors: Jabbour Elias , O’Brien Susan , Ravandi-Kashani Farhad , Cortes Jorge , Borthakur Gautam , Verstovsek Srdan , Garcia-Manero Guillermo , Giles Francis , Zanoria Lourdes , Davisson Jan , Issa Jean-Pierre , Kantarjian Hagop M.
Published: October 2019

Background The hypomethylating agents decitabine and azacitidine have significant anti-myelodysplastic (MDS)activity and have been FDA approved for the treatment of MDS and CMML. Experience in CMML has been minimal.

Study Aims Evaluate the efficacy of decitabine in CMML.

Study Group and Treatment Patients with CMML received decitabine 20 mg/m2 IV/1 hour daily x 5 or 2 alternative schedules, delivering 100 mg/m2/course every four weeks (Kantarjian, Blood 2006). Response was evaluated by the modified IWG criteria (Cheson, Blood 2006).

Results Nineteen patients were treated. Their median age was 66 years (range 44–82 years). Splenomegaly was present in 4 (21%); hemoglobin less than 10 g/dL in 10 (53%); thrombocytopenia less than 100 x 109/L in 12 (63%); WBC more than 40 x 109/L in 5 (26%); chromosomal abnormalities in 6 (32%); prior therapy for CMML in 9 (47%); marrow or peripheral blast 5% or more in 11 (58%). Overall, 13 patients (69%) achieved IWG responses: CR in 11 (58%), other HI in 2 (11%). Improvement of thrombocytopenia was noted in 7/8 patients (88%) with pretreatment platelets less than 50 x 109/L. Median survival was 19 months. Among 6 patients with pretreatment chromosomal abnormalities who achieved response, 1 (17%) had disappearance, and 2 had > 50% reduction of cytogenetic abnormalities. Extramedullary toxicities were minimal.

Conclusions Decitabine is active and safe in CMML. Future studies may combine decitabine with histone deacetylase inhibitors, chemotherapy (eg cytarabine, topotecan), or growth factors.
Expression of Proliferation Related Genes in Myelodysplastic Syndromes (MDS) and Secondary Acute Myeloid Leukemia (AML) Patients with Resistance to Low Dose Vidaza and Thalidomide.
Authors: Galili Naomi , Ebert Benjamin L. , Westervelt Peter , Wasserman Jessica , Lee Daniel , Raza Fatima Z. , Golub Todd R. , Raza Azra
Published: October 2019

Vidaza and Thalidomide were administered to 29 patients with MDS or AML. Vidaza was given at a dose of 75mg/kg subq X 5 days q28 days and Thalidomide starting at 50mg/day and increasing to 100mg. Therapy was well tolerated. Median age was 70 years, and there were 16 males. Two patients had RA, 2 RARS, 9 RAEB, 4 CMMoL, 10 AML and 2 Unknowns. According to IPSS, 1 had low, 7 had Int-1, 5 had Int-2 and 4 had high risk disease, and 2 unclassified and 10 had AML. Eleven patients had normal, 14 abnormal and 4 unknown cytogenetics. Hematologic improvement (HI) was seen in 14 and stable disease in 6 while 5 had disease progression. cDNA from pre-therapy BM mononuclear cells of 28/29 patients was hybridized to Affymetrix HG-U133AA microarrays and data was analyzed using the GenePattern software package. Marker genes were selected using ClassNeighbors algorithm with the signal-to-noise metric. When comparing resistant disease (RD) patients with HI (6) and others (2), of the 40 top marker genes over-expressed in the RD group, 21 are directly associated with cellular proliferation and can be divided into 3 groups. Group I (cell cycle) consisted of 9 genes; BUB1B, CCNA2, TMPO, CDC2, CCNB1, UBE2C, TTK, CDC20 and MCM5. Group II (replication repair) had 5 genes; TOP2A, RRM2, RFC3, FEN1, RNAH2A. Group III (spindle/chromosome structure) had 7; KIF2C, NUSAP1, CENPF, MK167, SPAG5, CENPA and STK6. Over-expression of these genes would indicate rapid cellular proliferation which may be an underlying cause of drug resistance. These results are especially striking since the treatment regimen consisted of a combination of two drugs, Vidaza and thalidomide, which purportedly act through different mechanisms. We conclude that the RD patients share a molecular signature which is also consistent with a demonstrated mechanism of cell cycle resistance, and underscores the future clinical usefulness of this methodology.
Erythroid Response (ER) Rates in Myelodysplastic Syndromes (MDS) Patients Treated with Epoetin Alfa (EPO) or Darbepoetin Alfa (DARB) Using International Working Group Response Criteria (IWGc): Comparative Meta-Analysis.
Authors: Mundle Suneel , Lefebvre Patrick , Duh Mei S. , Bourezak Ahmed , Yektashenas Behin , Moyo Victor
Published: October 2019

Background: EPO has been used in the treatment of refractory anemia in MDS patients for many years. Recently, with the identification of definitive patient selection criteria (low serum EPO [sEPO] levels and receipt of minimal transfusions) and standardization of ER rate evaluation methods using the IWGc, response to EPO therapy has been shown to be significantly enhanced in low/int-1 risk MDS patients. The present meta-analysis was conducted to compare ER rates (defined as major + minor response) of the two erythropoiesis-stimulating therapies (ESTs) currently available in the US, EPO and DARB, in MDS patients who were selected and evaluated for ER using the aforementioned uniform criteria.

Methods: A systematic review and data extraction of studies from PubMed and ASCO/ASH proceedings from 1990–2006 in MDS patients treated with EPO monotherapy and from 2003–2006 in those treated with DARB monotherapy was performed. Pooled estimates of ER rates were calculated in each group using random-effects meta-analysis methods, which incorporated both between- and within-group variations. Multivariate meta-regression analysis was further conducted to control for differences in baseline characteristics between the two groups.

Results: Nine EPO studies (N=619 patients, N evaluable for ER rate =589) and 8 DARB studies (N=442, N evaluable for ER rate =389) were identified. Baseline characteristics were comparable between the two groups with respect to age, gender, mean baseline hemoglobin, transfusion dependency rates, and proportion of patients with refractory anemia/refractory anemia with ringed sideroblasts. The mean baseline sEPO levels were significantly higher in the EPO group, compared to the DARB group (376: range 43–418; vs. 133: range 68–303 mU/mL, p=0.0026). The average initial weekly dose in the EPO studies was 47,851 (range: 30,000–80,000) Units, while that in the DARB studies was 176 (range: 100–315) mcg. There was no significant difference in the pooled estimate of the ER rates for either EST (57.6%: 95% CI; 45–70) for the EPO studies vs. (59.4%: 95% CI; 49–70) for the DARB studies, (p=0.8282). Further, as compared to the studies with standard doses of the two ESTs per week (EPO 30,000–40,000 Units; DARB ≤ 150 mcg), those using higher doses of EPO (60,000–80,000 Units) or DARB (>150 mcg) per week showed significantly higher ER rates (EPO- 47.8% vs. 63.3%, p<0.001; DARB −52.6% vs. 71.1%, p<0.001).

Conclusions: This current meta-analysis demonstrates that, with the use of standardized patient selection and response evaluation methods, the two clinically available ESTs yield comparable ER rates in MDS patients.
Clinical Activity of the Immunoconjugate CMC-544 in B-Cell Malignancies: Preliminary Report of the Expanded Maximum Tolerated Dose (MTD) Cohort of a Phase 1 Study.
Authors: Fayad Luis , Patel Hemant , Verhoef Gregor , Czuczman Myron , Foran James , Gine Eva , Rohatiner Ama , Smith Mitchell R. , Shapiro Mark , Advani Anjali
Published: October 2019

Introduction: CMC-544 is an antibody-targeted chemotherapy agent composed of a humanized antibody that specifically targets the CD22 antigen, conjugated to calicheamicin, a potent cytotoxic antitumor agent. Malignant cells of mature B-lymphocyte lineage express CD22, suggesting that CMC-544 may be useful for treating lymphomas of B-cell origin. A phase 1 dose-escalation trial of CMC-544 was performed at 14 European and US sites with 36 patients in the dose escalation portion and 48 in the expanded MTD portion. The MTD dose was 1.8 mg/m2 every 4 weeks. In the dose escalation phase the main toxicities observed were thrombocytopenia, asthenia, nausea, neutropenia, elevated liver function tests (LFTs) and anorexia. Grade 3–4 levels were only seen for thrombocytopenia, asthenia, neutropenia and LFTs (incidence of 40%, 13%, 9% and 9% respectively). Responses were seen in 8/22 (36%) patients (Advani A, et. al. Blood, abstract# 230, 2005:106). We now report the results of the expanded cohort at the MTD.

Patients and Methods: Relapsed/refractory lymphoma patients were treated at the 1.8 mg/m2 dose level every 4 weeks. In addition to safety data, preliminary efficacy data (assessed using the International Workshop to Standardize Response Criteria for NHL) were collected.

Results: As of July 2006, 48 patients were treated: median age 57 years (range 26–75); 51% females; 61% with ≥ 4 prior lines of therapy; 22 (46%) follicular lymphomas (FL) and 26 (54%) diffuse large B-cell lymphomas (DLBCL). Data were available on 48 patients evaluable for safety and 34 patients (19 FL and 15 DLBCL) evaluable for response. The overall safety profile was manageable; the most common drug-related adverse events (all grades) included thrombocytopenia (90%; the only bleeding noted was grade 1–2 epistaxis [12%]), asthenia (57%), nausea (39%), neutropenia (37%) and elevated levels of AST/SGOT (41%), ALT/SGPT (18%), alkaline phosphatase (27%) and bilirubin (18%). Grade 3–4 AEs that occurred with a frequency ≥ 10% included thrombocytopenia (57%) and neutropenia (29%). Responses in evaluable patients are shown in Table 1. The objective response rate was 69% and 33% for patients with FL and DLBCL, respectively.

Conclusions: CMC-544 exhibits effficacy against recurrent/refractory B-cell lymphomas, with the main toxicity being clinically manageable, self limited thrombocytopenia. These encouraging data support the continuing development of CMC-544.

Number (%) of Responses in Evaluable Patients: Response Follicular Lymphoma (n=19) DLBCL (n=15) ORR = Overall Remission Rate, (CR/CRu+PR) CR/CRu 6 (31.7) 2 (13.3) PR 7 (36.8) 3 (20.0) ORR 13 (68.5) 5 (33.3)
The Risk of Venous and Arterial Thrombosis in Hyperhomocysteinemic Subjects Is Caused by Elevated Factor VIII:C Levels.
Authors: Lijfering Willem M. , Veeger Nic J.G.M. , Brouwer Jan-Leendert P. , van de Poel Marlene H.W. , van der Meer Jan
Published: October 2019

Hyperhomocysteinemia is demonstrated as a risk factor for venous and arterial thrombosis. Experimental evidence suggests that its thrombogenic propensity results from endothelial dysfunction and injury followed by platelet and fibrin formation. However, lowering homocysteine concentrations with vitamin B6, B12 or folic acid has not resulted in a reduced risk of recurrent venous and arterial thrombosis in large prospective clinical trials. This suggests that hyperhomocysteinemia is a surrogate for another thrombophilic related specimen. As high factor VIII:C levels are associated with an increased risk of both venous and arterial thrombosis, and with endothelial injury, we hypothesize that hyperhomocysteinemia and factor VIII:C levels are closely related to each other. Therefore, we performed a study to assess the absolute risk of thrombosis in hyperhomocysteinemia and the effects of elevated factor VIII:C levels on this risk in a large cohort of families with hereditary (index) deficiencies of protein S, protein C or antithrombin. Hyperhomocysteinemia was defined as a fasting plasma homocysteine level above 18.5 μmol/l and factor VIII:C levels were elevated when higher than 150%. A total of 405 relatives were included. Median factor VIII:C levels in hyperhomocysteinemic relatives (n=26, 6%) were 169%, compared to 136% in normohomocysteinemic relatives (P=0.004) (Figure) and were more often elevated (65 vs. 38%, P=0.006). Other thrombophilic defects, including the index deficiencies, factor V Leiden and the prothrombin mutation were equally divided. Hyperhomocysteinemia was associated with an increased risk of venous and arterial thrombosis (relative risk’s 2.6 [1.3–4.8] and 3.7 [1.5–8.4)], respectively). Relatives with elevated factor VIII:C levels were also at risk; relative risk 2.3 (1.4–4.0) for venous thrombosis and 2.3 (1.0–5.1) for arterial thrombosis. After excluding all relatives with elevated factor VIII:C levels, relative risk for venous thrombosis and hyperhomocysteinemia dropped to 1.3 (0.2–9.8) and nil relatives had arterial thrombosis. These results suggest that hyperhomocysteinemia indeed is an epiphenomenon for elevated factor VIII:C levels and therefore homocysteine measurements can be omitted in risk assessment for venous and arterial thrombosis when factor VIII:C measurements are incorporated in thrombophilia screening.

Figure Figure
Immuno-Chemotherapy (IC) and Autologous Stem Cell Transplant (ASCT) for Untreated Patients (pts) with Mantle Cell Lymphoma (MCL): CALGB 59909.
Authors: Damon Lloyd E. , Johnson Jeff , Niedzwiecki Donna , Cheson Bruce D. , Hurd David Duane , Bartlett Nancy L. , Byrd John C. , Kelly Michael , Linker Charles , Canellos George P.
Published: October 2019

We evaluated the feasibility, safety, and effectiveness of an intense IC regimen including ASCT in pts with untreated MCL up to age 70. The primary end-point was 2-year progression free survival (PFS). A successful protocol would demonstrate a 2-year PFS of 50% (95% CI 37, 63%) or better. MCL was confirmed by central pathology review plus either compatible immunophenotype, cyclin D1 over-expression, t(11;14) or bcl-1/IgH rearrangement. Treatment consisted of 2 cycles (3 if marrow has >=15% MCL after 2 cycles) of rituximab (R), methotrexate 300 mg/m2 (M) and augmented CHOP (2 g/m2 cyclophosphamide [C]) followed by consolidation and stem cell collection with EAR (etoposide [E] 40 mg/kg, cytarabine [A] 2 g/m2 for 8 doses, R for 2 doses, and filgrastim 10 mcg/kg/day beginning day14 until completion of stem cell collection). EAR was followed by ASCT using CBV (carmustine [B] 15 mg/kg, E 60 mg/kg, and C 100 mg/kg) followed by R 6 and 7 weeks after ASCT. 79 pts were enrolled in 40 months: median age 57 (37–69); 79% male; 33% elevated serum LDH; 73% bone marrow (+); 5% cerebrospinal fluid (+). 1 pt never received protocol therapy, 1 pt came off study for an allogeneic stem cell transplant, and 7 pts never underwent ASCT. Stem cell collection was excellent, with a median CD34-cell dose of 12x106/kg in median of 1 apheresis. There were 2 treatment-related deaths. The complete and partial response rates were 62% (49,74%) and 27% (17, 40%), respectively. With a median follow-up of 27.5 months, the PFS is 75% (61, 84%) at 2 years and 56% (39, 69%) at 3 years (intention to treat analysis). The overall survival is 82% (68, 90%) at 3 years. Intense IC plus ASCT is feasible, safe, and effective in pts with MCL. These results are comparable to single institution outcomes. Maintenance bortezomib will be added to this regimen post-ASCT in a successor CALGB trial.
Does Rituximab Influence the Prognostic Effect of Germinal Centre Phenotype in Diffuse Large B-Cell Lymphoma?.
Authors: Nyman Heidi , Karjalainen-Lindsberg Marja-Liisa , Taskinen Minna , Berglund Mattias , Adde Magdalena , Amini Rose-Marie , Blomqvist Carl , Enblad Gunilla , Leppä Sirpa
Published: October 2019

PURPOSE: Germinal centre (GC) and non-GC phenotypes are predictors of outcome in diffuse large B-cell lymphoma (DLBCL), and can be used to stratify chemotherapy-treated patients into low- and high-risk groups. Our aim was to determine how combination of rituximab with chemotherapy influences GC-associated clinical outcome.

METHODS: 95 DLBCL patients treated with rituximab in combination with CHOP or CHOEP regimen (immunochemotherapy) were included in the study. The median follow-up time was 27 months. 107 patients previously treated with chemotherapy served as a historical control group. BCL-6, CD10, and MUM1 expression was analyzed immunohistochemically by means of identifying GC and non-GC phenotypes. In addition, BCL-2 expression was determined. Expression data was correlated with survival parameters.

RESULTS: Consistent with previous studies, chemotherapy-treated patients with GC phenotype displayed a significantly better overall survival (OS) than the non-GC group (70% vs 47% p=0.012). In contrast, GC-phenotype did not predict outcome in immunochemotherapy-treated patients. The OS for GC-group was 82% compared with 80% for those with non-GC phenotype (p=ns). Conversely, the relapse free survival (RFS) for patients with GC and non-GC phenotypes were 74% and 65% (p=ns), respectively. When the patients were grouped according to BCL-2 expression, survival rates among the group of BCL-2 negative patients tended to be better than in BCL-2 positive patients (OS, 94% vs 77%, p=0.097; RFS, 94% vs 66%, p=0.029).

CONCLUSIONS: Rituximab in combination with chemotherapy seems to interfere with the prognostic value of GC- and non-GC phenotypes in DLBCL. However, rituximab may have a positive impact on outcome among the patients with BCL-2 negativity.
Serum Levels and Pharmacokinetic of Rituximab in Bi-Weekly R-CHOP in Elderly Patients with DLBCL Treated in the RICOVER-60 Trial.
Authors: Reiser Marcel , Wenger Michael K. , Nickenig Christina , Peter N. , Kuhn Christian , Metzner Bernd , Theisohn Martin , Pfreundschuh Michael
Published: October 2019

Background: The combination of Rituximab and CHOP is considered the standard of treatment for DLBCL in elderly patients (pts) but there is hardly any data on the pharmacokinetic of Rituximab in aggressive lymphoma pts.

Objective: To study serum levels and pharmacokinetic properties of Rituximab when combined with dose dense CHOP-14 in elderly DLBCL patients

Methods: Blood samples of 18 pts were taken before and after Rituximab-infusion at each chemotherapy cycle. Additional samples were taken after the end of treatment and at the following timepoints: after 1 week, 1 month, 2 months, 3 months, 6 months and 9 months, respectively. Serum samples were taken within a maximum of 30 min, centrifuged at 1000 g for 10 min (room temperature) and stored at −20 degrees C. Batch samples were shipped to Xendo Lab., Groningen, The Netherlands, and analysed. The measured values were analysed by pharmakokinetic software MW/PHARM, version 3.50; (MediWare, 9800 AB Zuidhorn, The Netherlands) Additionally, a simulation of expected Rituximab-PK was performed taking into account 5 clinical trials, totalling 3777 observations in 264 patients mostly FL.

Results: Median (range) age of pts was 69 (61–79) years. 10/18 pts had advanced stage (III/IV), 4/18 pts had bulky disease. Pts received a median (range) dose of 683 (545–791) mg Rituximab/cycle. Response to treatment was 15/18 CR/Cru, 2/18 PR,1/18 PD. The median (range) of trough serum Rituximab-levels (μg/ml) before each cycle were: #1 0(0–0); #2 39(18–61); #3 74(47–109); #4 95(40–136); #5 113(55–157); #6 114(12–518); #7 125(72–207);#8 117(75–304). After therapy Rituximab-levels were: 163(68–248) at 1 week; 101(44–163) at 1 month, 55(11–122) at 2 months, 33(6–577) at 3 months; 5(0–103) at 6 months, 0.7(0–128) at 9 months. 7 pts were evaluable at 9 months with 4/7 pts showed detectable Rituximab-levels. Thus, actual serum levels detected fit exactly the model calculated from data of patients with FL. A preliminary PK analysis of Rituximab according to a two-compartment model showed a long median (range) serum half-life (t 1/2) of 765 h (543–1092).

Conclusion: In the dose dense bi-weekly regimen R-CHOP-14 Rituximab-serum levels increased slowly up to cyle 5. During cycle 5– 8 the serum Rituximab-levels reached a plateau and decreased constantly after the end of treatment with detectable levels even after 9 months and a long serum half-life. Increasing serum levels in the first four cycles are due to saturation kinetics of Rituximab. Based on this data the German High Grade NHL Study Group (DSHNHL) further investigates a dose escalation of Rituximab in the first cycles in order to improve treatment outcome.
Induction with Fludarabine, Cyclophosphamide and Rituximab Followed by Maintenance with Rituximab: Results of a Prospective Study in 75 Patients (LNHF-03).
Authors: Tomas Jose F. , Montalban Carlos , Martinez-Lopez Joaquin , Paz Jose , Canales Miguel , Diaz-Mediavilla Joaquin , Godoy Pedro Sanchez , de Sevilla Alberto Fdez , Penalver Javier , Caballero MDolores , Prieto Elena , Salar Jose , de Ona Raquel , de la Serna Javier
Published: October 2019

Objectives. To evaluate clinical efficacy, molecular response and safety following the use of a regimen of fludarabine (25 mg/m2 x 3 days) Cy (1g/m2 x 1 day) and Rituximab (375 mg/m2/ x 1 day) (FCR)x 6 cycles, followed by maintenance with rituximab (375 mg/m2/week/x 4 weeks every 6 months x 2 years). (LNHF-03 Study).

Patients and methods. Seventy-five (75) patients with a diagnosis of follicular NHL were included in the study between October 2004 and January 2006. The median age was 54 years (30–75). Twenty-one per cent (21%) of the patients had bulky disease. According to the FLIPI index: (0–1): 22.6%; 2: 40,3%; 3: 37,1%. Minimal residual disease (MRD): A clonal population on diagnosis was identified by means of the bcl–2/IgH major and minor rearrangement study by means of quantitative PCR rearrangements of the Igs by means of fluorescent PCR. Subsequently, samples were studied following the induction treatment and during the maintenance treatment. The analysis is performed in the 70 patients that completed the six induction regimens and were evaluated.

Results. 64/70 patients were given the six cycles planned (91%). The presence of persistent cytopenia limited the cycles given in the other 6 patients to 4 or 5. Three hundred and thirty-eight (338) adverse effects were documented in the 414 cycles given to the 70 patients evaluated, almost all of them being mild, and 26 severe (degree 3–4), and almost all the cases were neutropenias. Infectious complications were frequent and many were severe: herpes zoster (5), pneumonia (5), cerebral toxoplasmosis (1), aspergillosis (1), infection by CMV (2). One patient developed erythroleukaemia within the first year following completion of the treatment. Three patients died ( CMV + aspergillosis, pneumonia, erythroleukaemia). 86% of the patients reached CR following induction, 6% unconfirmed CR and 8% partial response. On diagnosis, a clonal population was identified in 63% (45/71) of the cases. Of these 45 patients, samples are available following induction treatment in 35 cases. The molecular disease was negativised in all the cases, except one case with persistence of positive bcl2/IgH (0.3% as opposed to diagnosis) and is in uncertain CR. Of the 34 cases with negative ER, 32 are in CR or uncertain CR and 2 PR.

Conclusions. The FCR regimen has proven to have a potent antitumoral activity in recently diagnosed follicular lymphoma patients with very high clinical (86% CR) and molecular (95%) responses. The immunosuppression caused is profound, with the appearance of opportunistic infections, and in some cases prolonged lymphopenias and neutropenias that call for future assessment and follow-up.
Combined Immuno-Chemotherapy Followed by Rituximab Maintenance Is an Effective Salvage Treatment after Prior Rituximab Containing Therapy: Results of a GLSG-Subgroup Analysis in Patients with Relapsed Indolent Lymphoma.
Authors: Dreyling Martin H. , Forstpointner Roswitha , Boeck Hans-Peter , Repp Roland , Wandt Hannes , Pott Christiane , Seymour John Francis , Metzner Bernd , Haenel Annette , Lehmann Tanja , Hartmann Frank , Einsele Hermann , Unterhalt Michael , Hiddemann Wolfgang
Published: October 2019

Background: Combined immuno-chemotherapy incorporating Rituximab with various chemotherapy regimens has resulted in increased response rates and prolonged progression-free and even overall survival in numerous trials. However, a constant relapse pattern has been observed even after such an optimized first line treatment.

Methods: In a subgroup analysis of the GLSG trial for relapsed indolent lymphoma (R-FCM followed by Rituximab maintenance), response and long term results of patients with previous Rituximab containing treatment were compared with the Rituximab naive control group. Induction comprised of 4 courses of chemotherapy with Fludarabine, Cyclophosphamide and Mitoxantrone (FCM) plus Rituximab. Patients responding with a complete (CR) or partial remission (PR) were randomized for observation only versus Rituximab maintenance (4 applications at month 3 and 9).

Results: 18 of 268 patients (arm A) with relapsed lymphoma had already previously received a R-containing regimen, the remainder 250 patients (arm B) were Rituximab naive. Overall response (arm A: 83 % vs. group B: 77%) and CR rate (arm A: 39% vs. arm B: 23%) were comparable in both subsets of study patients. Accordingly, progression-free survival after R-FCM was comparable in both subgroups of patients (median PFS in arm A: 15 months, arm B: 27 months) and overall survival as well. Especially, no disadvantage in the Rituximab pretreated patient group was detectable. 9 of the Rituximab pretreated patients responding to R-FCM induction therapy were randomized to maintenance treatment. After a median observation time of 20 months, 3 patients had relapsed and 6 patients remained in remission (3 of them for >20 months) resulting in a similar PFS after rituximab maintenance as in patients who were Rituximab naive.

Conclusion: This subgroup analysis strongly suggests that a Rituximab containing salvage therapy induction as well as maintenance is a highly effective treatment option even in patients who received Rituximab in first line therapy.
Favorable Results of Rituximab in Combination with CHOP in the Front-Line Treatment of Follicular Lymphoma Grade 3.
Authors: Fayad Luis , Overman Michael , Pro Barbara , McLaughlin Peter , Samaniego Felipe , Younes Anas , Romaguera Jorge Enrique , Hagemeister F.B. , Kwak Larry , Rodriguez Maria Alma
Published: October 2019

Background: Follicular lymphoma grade 3 has a natural history that is more akin to that of diffuse large B-cell lymphoma. The addition of rituximab to standard CHOP has resulted in improved response and survival in diffuse large B-cell lymphoma. Information about outcomes in follicular lymphoma grade 3 is lacking.

Methods: A single institution retrospective review of patients with follicular grade 3 lymphoma evaluated at the UTMDACC from 1999 to 2004. Patients were located from the UTMDACC lymphoma database. All patients were initially treated with R-CHOP.

Results: Forty-five patients were identified: 51% male, 47% ≥60 years, and 87% follicular grade 3b. The LDH was elevated in 24%, ECOG performance status was >1 in 2%, and >1 site of extranodal involvement was present in 10%. Stage distribution was 11% stage I, 11% stage II, 42% stage III, and 36% stage IV, bulky disease (>7cm) was present in 11%, and B symptoms occurred in 13%. Beta-2 microglobulin was elevated in 57% with values >3 μg/dL in over 50%. IPI distribution was: 46% IPI Low, 38% LI, 11% IH, and 4% IPI High. Overall response rate was 100% with 96% complete responses. Relapse rate by IPI category was 24% for Low IPI, 18% for IPI LI, and 40% for IPI IH, and 100% for the two patients with High IPI. With median follow-up of 33 months, three year failure-free survival (FFS) is 73% (95% CI: 59 to 87%). One patient died (2%) with an overall survival (OS) at three years of 97% (95% CI: 93 to 100%).

Conclusion: The addition of rituximab to CHOP provided a high response rate and excellent early survival in this group of mostly good prognosis patients. Relapses were still seen; longer follow-up is needed.
FMNL-1 (Formin-Like 1) Gene in Chronic Lymphocytic Leukemia (CLL) Is Overexpressed in Young Patients with Adverse Prognostic Factors and Poor Outcome.
Authors: Calpe Eva , Favaro Patricia M.B. , Crespo Marta , Baptista Maria Joao , Muntañola Ana , Giné Eva , Saad Sara , Montserrat Emili , Bosch Francesc
Published: October 2019

Prognosis of patients with CLL has been traditionally assessed by using clinical parameters. Although useful, such parameters are a mere reflection of the biological diversity of CLL. In this regard, the mutational status of VH genes or ZAP-70 expression separates CLL into two clinical forms with different presenting features and outcome. Formins are multidomain proteins characterized by the presence of two conserved prolin-rich regions, namely formin homology 1 and 2. These proteins are implicated in a wide range of processes, including regulation of the cytoskeleton and in the regulation of the signal for cell survival. Formin is normally expressed in spleen, lymph node, and bone marrow cells, and it has been recently found to be overexpressed in T-cell lymphomas. The aim of this study was to analyze the expression of FMNL-1 in normal B-cell subsets and in a series of 73 patients (median age, 59 years; male/female 40/33; Binet A: 90.2%) with CLL. FMNL-1 expression was analyzed by Western Blot in separate subpopulations and by quantitative RT-PCR using expression in Jurkat as baseline. Among normal lymphocytes, FMNL-1 was only expressed in memory (CD19+CD27+) B-cells and in T-cells. In CLL cases with a low percentage of T-cells, mean of FMNL-1 expression was 2.18 AU (SD, 1.01 AU). Using an arbitrary cut-off of 3.2 AU, cases with increased expression of FMNL-1 were associated with a younger age at diagnosis (< 50 yrs), elevated lymphocyte count, high serum β2-microglobulin (β2-m) levels, increased ZAP-70 and CD38 expression, shorter time to progression, and shorter survival as compared to cases with low FMNL-1 expression. No relationship was observed with genetic abnormalities (table). In summary, among B-cell lymphoproliferative disorders, FMNL-1, a gene that regulates cell survival, is found only in CLL and its overexpression correlates with adverse clinical and biological parameters, particularly in young patients.

Variable FMNL-1 normal FMNL-1 increased p value Age < 50 yrs 15% 86% 0.0001 Lymphocyte count > 50.000/ μL 14% 57% 0.018 Increasedβ2-microglobulin 17% 66% 0.038 CD38 > 30% 28% 100% 0.001 ZAP-70≥20% 50% 100% 0.014 Time to progression (median) 5.1 yrs 0.5 yrs 0.003 Survival (median) 16.4 yrs 8.5 yrs 0.009
Prolonged Survival of Chronic Lymphocytic Leukemia Cells Induced by Sphingosine 1-Phosphate Is Mediated by the G Protein-Coupled Receptor S1P1 and Involves Erk/MAP Kinase, but Not Akt Signaling.
Authors: Seitz Gabriele , Yildirim Sedat , Boehmler Andreas , Kanz Lothar , Mohle Robert
Published: October 2019

Sphingosine 1-phosphate (S1P) is a bioactive lysophospholipid mediator which is generated by the hydrolysis of sphingomyelin and released predominantely by activated platelets but also by other cells upon activation. In normal blood plasma it is found in nanomolar to micromolar concentrations. By qualitative and quantitative (TaqMan) RT-PCR, mRNA expression of the S1P receptor S1P1 was detected in CLL cell lines (EHEB, MEC-1) and in all samples of primary chronic lymphocytic leukemia (B-CLL) cells, analyzed after isolation with a CD19 antibody and magnetic cell sorting. mRNA expression of other S1P receptors was less consistently found. When primary CLL cells were cultured in serum-free medium (RPMI 1640 supplemented with fatty acid-free BSA), viability decreased starting from 3 days of culture and was completely lost after 1–2 weeks depending on the individual sample. However, when the cultures were supplemented with S1P (1 nM - 5 μM) the number of viable cells was increased up to 8-fold in a time- and dose-dependent manner as measured using the WST-1 reagent. Moreover, S1P induced a rapid (maximum at 1 min) phosphorylation of the p44/42 Erk/MAP kinase in CLL cell lines and even more prominent in primary CLL cells. The time- and dose dependent phosphorylation was blocked by pretreatment (24h) of the cells with FTY720 (10–100 nM), a compound that binds predominantly to S1P1 and induces loss of cell surface expression by receptor internalization. FTY720 also abrogated the prolonged survival of CLL cells in cultures supplemented with S1P, demonstrating that both the Erk/MAP kinase phosphorylation and the consecutive increased survival were mediated by the S1P1 receptor. Activation of MAP kinase was also inhibited by pertussis toxin, which selectively blocks Gi proteins, the only class of G proteins known to be coupled to S1P1. Phosphorylation of Akt was not observed in CLL cell lines and primary CLL cells after stimulation with S1P. B-CLL cell lines showed a pronounced chemotactic response to S1P at low concentrations. In primary CLL cells however, S1P did neither induce phosphorylation of the focal adhesion kinase (FAK) nor chemotaxis. We conclude that S1P, constitutively present in the blood, prolongs survival of CLL cells involving the Erk/MAP kinase pathway. Since compounds that induce S1P1 receptor internalization like FTY720 are already used as immunosuppressants in humans, our results suggest inclusion of these drugs also in the treatment of B-CLL, where they may potentiate the effect of conventional antiproliferative therapy.
Activation by CD40 Ligand Shifts Bcl-2 Family Protein Expression from Death-by-Default to Survival-by-Default.
Authors: Willimott Shaun , Baou Maria , Huf Sarah , Wagner Simon
Published: October 2019

CLL cells survive and accumulate in vivo and yet leukaemic cells isolated from the peripheral blood rapidly die. We have used mouse fibroblast L-cells transfected with CD40 ligand (CD40L) in combination with IL-4 to reproduce some features of the in vivo microenvironment, which is essential for CLL cell proliferation. As anticipated we found that leukaemic cells survive and proliferate in this system. Also CD23 expression increases, but unexpectedly CD38 expression increased to varying degrees in all patients and CD5 expression decreased. Changes in CD23 and CD38 have been reported in CLL cells in lymph nodes and support the concept that we are able to some extent reproduce the in vivo microenvironment. Next we carried out a systematic analysis of Bcl-2 family proteins by Western Blot analysis. On tissue culture plastic and non-transfected L-cells Bcl-2 is the predominant pro-survival protein. The activator BH3-only protein Bim is expressed together with the sensitiser BH3-only Puma. Both pro-apoptotic Bak and Bax are expressed. By contrast on CD40L-L cell with IL-4 culture Bcl-2 expression decreases and the predominant prosurvival protein is Bcl-xL with a contribution from Mcl-1. The activator BH3-only protein is Bid rather than Bim and the sensitiser BH3 only proteins Puma, Noxa and Bad are expressed. Levels of Bak are significantly decreased but Bax expression is unchanged. Overall it appears that there is an excess of pro-survival Bcl-xL on CD40L-L cells/IL-4 with decreased amounts of Pro-apoptotic Bak. These findings are important when considering the place of treatments directed at pro-survival Bcl-2 family proteins. Large amounts of Bcl-xL is a reservoir to soak up the reduced amount of Bak suggesting that whilst Bcl-2 antagonists will tip the balance towards apoptosis in CLL cells activated by CD40L/IL-4 they are unlikely to be sufficient to cause cell death. However, leukaemic cells in the peripheral blood are dependent on Bcl-2 and have increased amounts of Bak. In this situation reduction in Bcl-2 will release sufficient Bak to cause apoptosis.
B-CLL Antibodies Comprised of Stereotypic VH1-69, D3-16, and JH3 Rearrangements Immunoprecipitate Cellular Protein(s).
Authors: Chu Charles C. , Wang Xiao Bo , Hatzi Katerina , Catera Rosa , Meffre Eric , Herve Maxime , Allen Steven L. , Rai Kanti R. , Chiorazzi Nicholas
Published: October 2019

B-cell chronic lymphocytic leukemia (B-CLL) is typically due to a clonal expansion of B-lymphocytes that can be divided into two classes based on the mutation status of the variable region of the antibody produced by the leukemic clone. Mutated B-CLL (antibody with ≥ 2% mutation) is associated with longer survival than unmutated B-CLL. Segregation of patients based on antibody mutation suggests a functional role for antigen binding to antibody in this disease. Furthermore, many different subsets of B-CLL antibodies have very similar CDR3 sequences, suggesting some common antigen reactivity. Subset I contains rearrangements comprised of an unmutated sequence encoded by VH1-69, D3-16, and JH3 with a nearly identical HCDR3 region. Antibodies from this subset strongly bind cytoplasmic structures in HEp-2 cells by immunofluorescence and are polyreactive against multiple autoantigens (ssDNA, dsDNA, insulin) by ELISA. In contrast, subset VI, which comprises a mutated sequence encoded by VH3-21 and JH6 resulting in a nearly identical HCDR3 that differs from that of subset I, does not bind HEp-2 cells and is not polyreactive. Because the subset I B-CLL antibodies were reactive with HEp-2 cells, we prepared HEp-2 cell extracts in order to isolate the cellular antigen recognized by these antibodies. Antibodies from both subset I (patients CLL068 and 258) and VI (patient CLL412) were recombinantly overexpressed and purified as human IgG1s. Antibody was bound to protein G agarose beads and incubated with cell extracts overnight. Bound cellular antigen was immunoprecipitated, washed, eluted, run on SDS-PAGE, and stained with Coomassie Blue. Both CLL068 and 258 antibodies immunoprecipitated protein bands at about 45 and 200 kDa. These bands have been isolated and are undergoing microsequencing analysis. In contrast, CLL412 did not immunoprecipitate any protein bands under the same conditions. Thus, unmutated subset I CLL antibodies react with common cellular protein(s), whose identity may be useful in understanding B-CLL.
Reduced Rates of B Cell Proliferation in Chronic Lymphocytic Leukemia.
Authors: Defoiche Julien , Debacq Christophe , Asquith Becca , Zhang Yan , Burny Arsène , Bron Dominique , Lagneaux Laurence , Macallan Derek , Willems Luc
Published: October 2019

Whether chronic lymphocytic leukemia (CLL) represents latent or proliferating disease has been intensively debated. Whilst the dogma that CLL results from accumulation of dormant lymphocytes is supported by the unresponsiveness of leukemic cells to antigens and polyclonal activators, recent in vivo kinetic measurements show that B-lymphocytes do divide at significant rates in CLL. However, B cell kinetics were not compared between CLL patients and healthy controls so it was not possible to ascertain to what extent lymphocyte kinetics were aberrant in CLL.

We compared proliferation rates of B- and T-lymphocytes in CLL patients and healthy controls, using a pulse-chase approach based on incorporation of deuterium from 6,6-2H2-glucose into DNA. We found dramatically reduced in vivo rates of CD3−CD19+ cell proliferation in CLL compared with controls (mean 0.47 versus 1.66 %/day respectively, P=0.001), equivalent to an extended half-life of circulating B-cells (147 days versus 42 days). Labeled (dividing) CD3−CD19+ cells had death rates similar to the healthy controls (2.29 versus 3.55 %/day, P=0.495). Despite such aberrant B-cells kinetics, T-cell proliferation was unaffected by CLL (1.77 versus 1.40 %/day, P=0.488). We conclude that, B-cell proliferation rates are reduced in leukemic patients compared to healthy subjects and that most circulating CD3−CD19+ cells are quiescent, long-lived cells.
Distinct Phenotype of Chronic Lymphocytic Leukaemia (B-CLL) Cells Engrafting in NOD/SCID Mice.
Authors: Ebeling Peter , Durig Jan , Grabellus Florian , Seeber Siegfried , Duehrsen Ulrich , Moritz Thomas
Published: October 2019

We have previously reported reliable engraftment of CD19/CD5/CD23/CD45 positive human B-CLL cells in the spleens of NOD/SCID mice (NSS-CLL cells) following transplantation of human peripheral blood-derived B-CLL (PB-CLL) cells. On histology, in the majority of the samples NSS-CLL cells formed focal aggregates which also contained various amounts (range:0–50%) of human CD45/CD3+ T cells (ASH 2005; #52). We now have further characterized these NSS-CLL cells. In our model 1x10^8 MNC are injected i.v. plus i.p. and engraftment in the spleen and other organs is analyzed after 4 (−8) weeks by flow cytometry and immunohistochemistry. In 22 CLL samples investigated in a total of 64 NOD/SCID mice 1.6±0.4x106 NSS-CLL cells representing 10.5±2.3% of total spleen cells were recovered with recovery highly reproducible on repetitive experiments (3.0 vs. 2.0; 1.6 vs. 1.5; 0.2 vs. 1.1; 0.0 vs. 0.0x106 NSS-CLL cells; n=4). In contrast to the low proliferative status reported for human PB-CLL, considerable proliferation was observed in NSS-CLL cells. Following the application of BrdU (1mg/6g d5 i.p., 1mg/ml added to drinking water d6–13; 20–27) BrdU positive NSS-CLL cells ranged from 5.3–25.0% (n=5). Immunohistological analysis showed positive staining for the proliferation marker Ki67 in 18.5±3.5% of NSS-CLL cells. In addition, markers related to proliferation/activation of B-CLL proliferation center cells such as survivin (mean MFI: 5.5±0.8 vs. 2.3±0.2; p=0.028; n=6) and CD100 (semaphorin 4d; mean MFI: 3.3±0.5 vs. 1.1±0.2; p=0.043; n=5) were significantly up-regulated in NSS-CLL vs. PB-CLL cells. When NSS-CLL cells were characterized utilizing additional adhesion/migration, differentiation and/or activation markers, only a subset of NSS-CLL cells (15.7±8.2%, n=6) expressed chemokine coreceptor (CCR) 7, while PB-CLL cells uniformly showed high CCR7 expression. Also CCR5, a migration marker in the context of non-Hodgkin lymphomas, consistently was demonstrated on a fraction of NSS-CLL cells (8.1±3.0%; n=6), whereas here no CCR5 expression was found on PB-CLL cells. No expression of human CD10, CD11c, CD127 or CD49d was detected in B-CLL cells derived from either PB- or NSS-CLL cells, while both sources, as expected, uniformly stained positive for CD40. In summary, NSS-CLL exibit a distinct phenotype as compared to PB-CLL cells and show considerable similarity to B-CLL cells in the proliferation centers of involved human tissues. For these cells not only proliferation but also increased expression of survivin and CD100 as well as down-regulation of CCR7 have been described. Therefore, we think that our model may represent a novel tool to further elucidate biological properties of human B-CLL and, in addition, may serve to devise and evaluate novel treatment strategies.
The Combination of Alemtuzumab [Continuous Intravenous Infusion (civ) Followed by Subcutaneous Injection (sc)] Plus Rituximab Has Activity in Patients (pts) with Relapsed Chronic Lymphocytic Leukemia (CLL).
Authors: Faderl Stefan , Ferrajoli Alessandra , Wierda William G. , O’Brien Susan , Ravandi-Kashani Farhad , Fayad Luis , Thomas Deborah , Kantarjian Hagop , Browning RN Mary L. , Keating MB Michael J.
Published: October 2019

Available data suggest enhanced activity of monoclonal antibodies in combinations. In a previous study of iv bolus alemtuzumab plus rituximab in relapsed CLL, we have shown that the combination is safe and produces a response rate of 54% following a 4-week treatment period (Blood101: 3413, 2003). As questions remain about the optimal dose, schedule and route of administration of alemtuzumab, we have been exploring alemtuzumab by civ followed by sc injections in combination with rituximab in pts with relapsed/refractory CLL who express both CD20 and CD52. Forty-five pts have been enrolled of whom 32 pts are evaluable (30 CLL, 1 CLL/SLL, 1 MZL). Six pts are too early and 7 are inevaluable [withdrawal of consent prior to treatment start (2), ineligible (2), removal from study and continuation of therapy with home physician (3)]. All pts received alemtuzumab 15mg by civ over 24 hours daily x 6 days on days 2–7 of the first course followed by 30mg sc twice weekly on days 3 and 5 of weeks 2 to 4 (one course). Rituximab was given at 375mg/m2 iv on day 1 followed by 500mg/m2 on days 8, 15, and 22. Maximum duration of therapy was 3 courses. Pts received standard premedication with acetaminophen and an antihistamine, and antiinfective prophylaxis with TMP/SMX and valacyclovir. CMV antigenemia was tested before and after completion of each course. The median age was 59 yrs (range 39–78), the median number of prior therapies was 3 (1–8), and the median b2M 3.8 mg/dL (1.7–13.6). Seventeen pts (53%) had Rai stage ≥ 3 disease. Eighteen pts (56%) were refractory to fludarabine and alkylators. All pts had prior rituximab; only 4 (13%) had received alemtuzumab. Sixteen pts (50%) responded [8 CR (25%), 1 PRn (3%), 7 PR (23%)] following one course. Response by site: peripheral blood 24/26 (92%), marrow 22/32 (69%), lymph nodes 13/26 (50%) and liver/spleen 8/9 pts (89). The combination was well tolerated with no unexpected toxicities. Most non-hematologic AEs were infusion-related and ≤ grade 2 by NCI toxicity criteria. Fevers and chills occurred in up to 56% of pts, fatigue in 34%, skin rashes in 19%, nausea in 16%, myalgias in 9%, and diarrhea in 6%. Although toxicites were more frequent with civ alemtuzumab than during the s.c. injections (with the exception of skin rashes), civ alemtuzumab was well tolerated. CMV reactivation occurred in 7 pts (22%) and was the most common infectious complication. In conclusion, the combination of civ/sc alemtuzumab plus rituximab has activity in patients with relapsed and refractory CLL. Most responses occurred after 4 weeks of therapy only. The combination is well tolerated. Side effects are predictable and manageable.