Quality of life assessment in diffuse large B-cell lymphoma (DLBCL) in REFLECT: a prospective, non-interventional, multicenter, German study, assessing Sandoz rituximab in combination with CHOP.

Health-related quality of life (HRQoL) data are important indicators of health status in patients with lymphoma. The objective of this analysis was to assess the impact of treatment with Sandoz rituximab plus cyclophosphamide, doxorubicin, vincristine, and prednisone (R-CHOP) on HRQoL in treatment-naïve adult patients with diffuse large B-cell lymphoma (DLBCL) included in the prospective, real-world REFLECT study. REFLECT is the first prospective study to assess HRQoL in patients with DLBCL treated with a rituximab biosimilar. HRQoL was assessed via the patient-reported European Organization for Research and Treatment of Cancer Core Quality of Life questionnaire at baseline, mid-treatment (month 3), end of treatment (month 6), and follow-up (months 9 and 12). Subgroup analyses were performed to evaluate the influence of baseline characteristics on HRQoL, and associations between baseline HRQoL and treatment response. HRQoL was assessed in 169 patients. Mean global health status score remained stable from baseline (54.8) to mid-treatment (month 3; 54.7), before steadily improving through to end of treatment (month 6; 61.4), and follow-up month 9 (64.9) and month 12 (68.8). Similar trends were observed across most functional and symptom subscales. Higher cognitive, physical, or role functioning, and less appetite loss, diarrhea, fatigue, or pain at baseline, were all associated with an improved likelihood of reaching a complete versus partial response at the end of treatment. Overall, these findings confirm the HRQoL benefits of R-CHOP therapy in treatment-naïve adult patients with DLBCL, and suggest that baseline HRQoL may be predictive of treatment response.

REFLECT: prospective multicenter non-interventional study evaluating the effectiveness and safety of Sandoz rituximab (SDZ-RTX; Rixathon®) in combination with CHOP for the treatment of patients with previously untreated CD20-positive diffuse large B-cell lymphoma.

Background: REFLECT is the first prospective study of Sandoz biosimilar rituximab (SDZ-RTX) in patients with diffuse large B-cell lymphoma (DLBCL). Objective: To evaluate the 2-year effectiveness and safety of SDZ-RTX as first-line treatment for DLBCL. Design: Real-world, multicenter, open-label, single-arm, non-interventional, post-approval study of SDZ-RTX in combination with cyclophosphamide, doxorubicin, vincristine, and prednisone (R-CHOP) in patients with treatment-naïve CD20­positive DLBCL. Methods: Treatment-naïve, CD20-positive adult patients (⩾18 years) with DLBCL eligible for therapy with R-CHOP were treated with SDZ-RTX-CHOP every 2 or 3 weeks for 6-8 cycles. The effectiveness of SDZ-RTX was measured by the complete response (CR) rate at the end of R-CHOP treatment, as assessed by the treating physician. Progression-free survival (PFS) was assessed at 24 months. Results: A total of 169 patients [52.1% female, median (range) age 70 (24-94) years] with DLBCL were included in the full analysis set. At baseline, 19.5% and 24.3% of patients had Ann Arbor disease stage III or IV, respectively, and most patients (80.5%) had Eastern Cooperative Oncology Group Performance Status of 0 or 1. A total of 100 (59.2%) patients completed the 24-month observation period. In total, 110 [65.1%; 95% confidence interval (CI): 57.4-72.3] patients achieved CR as best response and 50 (29.6%; 95% CI: 22.8-37.1) patients achieved partial response. Overall best response rate was 94.7% (95% CI: 90.1-97.5). One-year PFS was 84.9% (95% CI: 78.2-89.6), while 2-year PFS was 78.5% (95% CI: 70.9-84.4); median PFS was not reached within the observational period. A total of 143 (84.6%) patients experienced ⩾1 adverse event, 53 (31.4%) of which were suspected to be related to study drug. Conclusion: This real-world, 2-year study reconfirms that first-line treatment of CD20-positive DLBCL with R-CHOP using SDZ-RTX is effective and well tolerated. Registration: N/A.

REFLECT: A study evaluating Sandoz biosimilar rituximab (Rixathon ^® ) in combination with CHOP for the treatment of patients with previously untreated diffuse large B-cell lymphoma Why was this study done? • Biosimilars are biologic medicines that are highly similar to a reference biologic medicine that is already approved and has been used in patients for several years. • The REFLECT study was the first study of a biosimilar medicine (Sandoz biosimilar rituximab) in patients with a type of lymphatic cancer called diffuse large B-cell lymphoma (DLBCL). What did the researchers do? • Sandoz biosimilar rituximab was given as part of the standard treatment (cyclophosphamide, doxorubicin, vincristine, and prednisone; R-CHOP) in patients with DLBCL who had not received treatment before. • The researchers aimed to evaluate how well Sandoz biosimilar rituximab worked over a 2-year period. • The researchers also aimed to look at the safety of Sandoz biosimilar rituximab. • Patients with DLBCL had to be ⩾18 years of age, in need of treatment, and were classed as suitable for treatment with R-CHOP by their doctor. • Patients were treated with R-CHOP including Sandoz biosimilar rituximab every 2 or 3 weeks for 6­8 cycles. What did the researchers find? • A total of 169 patients with DLBCL were included in the study. • Just over half (52%) were female and the average age was 67 years. • Nearly 6 out of 10 (59%) patients completed the 2-year study. • More than 6 out of 10 (65%) patients achieved complete response and 3 out of 10 (30%) achieved partial response. • The overall response rate was 95%. • One-year progression-free survival was 85%, and 2-year progression-free survival was 79%. • Regarding safety, 85% of patients experienced at least one adverse event; just over 3 out of 10 (31%) of these were suspected to be related to the study drug. What do the findings mean? • This 2-year study shows that R-CHOP including Sandoz biosimilar rituximab is effective and well tolerated as the first treatment given to patients with DLBCL.

Pseudotyped recombinant adeno-associated viral vectors mediate efficient gene transfer into primary human CD34(+) peripheral blood progenitor cells.

BACKGROUND AND AIMS: Because of their pluripotency, human CD34(+) peripheral blood progenitor cells (PBPC) are targets of interest for the treatment of many acquired and inherited disorders using gene therapeutic approaches. Unfortunately, most current vector systems lack either sufficient transduction efficiency or an appropriate safety profile. Standard single-stranded recombinant adeno-associated virus 2 (AAV2)-based vectors offer an advantageous safety profile, yet lack the required efficiency in human PBPC. METHODS: A panel of pseudotyped AAV vectors (designated AAV2/x, containing the vector genome of serotype 2 and capsid of serotype x, AAV2/1-AAV2/6) was screened on primary human granulocyte-colony-stimulating factor (G-CSF)-mobilized CD34(+) PBPC to determine their gene transfer efficacy. Additionally, double-stranded self-complementary AAV (dsAAV) were used to determine possible second-strand synthesis limitations. RESULTS: AAV2/6 vectors proved to be the most efficient [12.8% (1.8-25.4%) transgene-expressing PBPC after a single transduction], being significantly more efficient (all P<0.005) than the other vectors [AAV2/2, 2.0% (0.2-7.3%); AAV2/1, 1.3% (0.1-2.9%); others, <; 1% transgene-expressing PBPC]. In addition, the relevance of the single-to-double-strand conversion block in transduction of human PBPC could be shown using pseudotyped dsAAV vectors: for dsAAV2/2 [9.3% (8.3-20.3%); P<0.001] and dsAAV2/6 [37.7% (23.6-61.0%); P<0.001) significantly more PBPC expressed the transgene compared with their single-stranded counterparts; for dsAAV2/1, no significant increase could be observed. CONCLUSIONS: We have shown that clinically relevant transduction efficiency levels using AAV-based vectors in human CD34(+) PBPC are feasible, thereby offering an efficient alternative vector system for gene transfer into this important target cell population.

A combination of granulocyte-colony-stimulating factor (G-CSF) and plerixafor mobilizes more primitive peripheral blood progenitor cells than G-CSF alone: results of a European phase II study.

BACKGROUND AIMS: Previous studies in xenograft models have shown that human peripheral blood progenitor cells (PBPC) mobilized with the CXCR4 antagonist plerixafor (AMD3100) have a higher bone marrow (BM) reconstitution potential than granulocyte-colony-stimulating factor (G-CSF)-mobilized PBPC. METHODS: PBPC obtained during G-CSF-supported mobilization before and after a supplementary administration of AMD3100 from patients with multiple myeloma and non-Hodgkin's lymphoma (n=15; phase II study) were investigated for co-expression of primitive and lineage-associated markers, their proliferative activity in vitro and repopulation potential after clinical transplantation. RESULTS: A significant increase in primitive CD34+ CD38(-) cells was observed in intraindividual comparisons of all patients after administration of G-CSF+AMD3100 (peripheral blood: median 8-fold, range 2,4-fold - 39-fold) compared with G-CSF alone. Using a long-term culture-initiating cell assay, this increase was confirmed. After transplantation of G-CSF+AMD3100-mobilized PBPC, the time to leukocyte reconstitution > 1 x 10(3)/microL and platelet reconstitution > 2 x 10(4)/microL was 14 (10-19 days) and 13 days (10-15 days), respectively. A complete and stable hematologic reconstitution (platelets > 1.5 x 10(5)/microL) was observed in 91% of all patients within 35 days. CONCLUSIONS: An additional application of AMD3100 to a standard G-CSF mobilization regimen leads to a significant increase in primitive PBPC with high repopulation capacity.

Combination treatment of imatinib-sensitive and -resistant BCR-ABL-positive CML cells with imatinib and farnesyltransferase inhibitors.

BACKGROUND: Resistance to imatinib monotherapy frequently emerges in advanced stages of chronic myelogenous leukemia (CML), supporting the rationale for combination drug therapy. In the present study, the activities of the farnesyltransferase inhibitors (FTIs) L744,832 and LB42918, as single agents and in combination with imatinib, were investigated in different imatinib-sensitive and -resistant BCR-ABL-positive CML cells. MATERIALS AND METHODS: Growth inhibition of the cell lines and primary patient cells was assessed by MTT assays and colony-forming cell assays, respectively. Drug interactions were analyzed according to the median-effect method of Chou and Talalay. The determination of apoptotic cell death was performed by annexin V/propidium iodide staining. RESULTS: Combinations of both FTIs with imatinib displayed synergism or sensitization (potentiation) in all the cell lines tested. In primary chronic phase CML cells, additive and synergistic effects were discernible for the combination of imatinib plus L744,832 and imatinib plus LB42918, respectively. Annexin V/propidium iodide staining showed enhancement of imatinib-induced apoptosis with either drug combination, both in imatinib-sensitive and -resistant cells. CONCLUSION: The results indicated the potential of L744,832 and LB42918 as combination agents for CML patients on imatinib treatment.

Myeloablative conditioning in myelofibrosis using i.v. treosulfan and autologous peripheral blood progenitor cell transplantation with high doses of CD34+ cells results in hematologic responses - follow-up of three patients.

Autologous transplantation after myeloablation for myelofibrosis with myeloid metaplasia provides a palliative therapy with a long term relief of symptoms. We have transplanted three patients with more than 5 x 10(6) CD34+ cells/kg body weight after myeloablation with treosulfan (total dose 42 g/m(2)) with a 18 months follow-up. Two of the patients had symptomatic splenomegaly and severe anemia. One patient had symptomatic splenomegaly and thrombocytopenia (< 100x10(9)/L). Granulocyte colony-stimulating factor-supported peripheral blood progenitor cell mobilization and collection was not associated with increased toxicity. Following transplantation we observed a prolonged reconstitution period of 28-38 days without fever or severe mucositis. All patients became free of erythrocyte transfusions or recovered to normal thrombocyte counts. There was a significant reduction of max. spleen size in one patient. We conclude that myeloablation with treosulfan and autologous PBPCT in these three patients with myelofibrosis was safe and useful.

Level of CD 20-expression and efficacy of rituximab treatment in patients with resistant or relapsing B-cell prolymphocytic leukemia and B-cell chronic lymphocytic leukemia.

Rituximab (IDEC C2B8) is a chimeric human-mouse monoclonal anti-CD20 antibody that has been proven to be effective for the treatment of patients with CD20 positive leukemia and lymphoma. The level of CD20-expression in patients with B-cell chronic lymphocytic leukemia (B-CLL) is low in comparison to other B-cell lymphomas and normal B-cells. Previous experience with rituximab treatment in small series of patients with B-CLL suggest that it is less effective in B-CLL than in follicular lymphomas. We analyzed the correlation between CD20-expression level and efficacy of rituximab treatment in eight patients with refractory or relapsed B-CLL and two patients with B-cell prolymphocytic leukemia (B-PLL). We could not identify any correlation between CD20-expression and efficacy of rituximab treatment.

In vitro testing of drug combinations employing nilotinib and alkylating agents with regard to pretransplant conditioning treatment of advanced-phase chronic myeloid leukemia.

PURPOSE: The prognosis of patients with advanced-phase chronic myeloid leukemia (CML) remains dismal despite the availability of targeted therapies and allogeneic stem cell transplantation (allo-SCT). Increasing the antileukemic efficacy of the pretransplant conditioning regimen may be a strategy to increase remission rates and duration. We therefore investigated the antiproliferative effects of nilotinib in combination with drugs that are usually used for conditioning: the alkylating agents mafosfamide, treosulfan, and busulfan. METHODS: Drug combinations were tested in vitro in different imatinib-sensitive and imatinib-resistant BCR-ABL-positive cell lines. A tetrazolium-based MTT assay was used for the assessment and quantification of growth inhibition after exposure to alkylating agents alone or to combinations with nilotinib. Drug interaction was analyzed using the median-effect method of Chou and Talalay, and combination index (CI) values were calculated according to the classic isobologram equation. RESULTS: Treatment of imatinib-sensitive, BCR-ABL-positive K562 and LAMA84 cells with nilotinib in combination with mafosfamide, treosulfan, or busulfan resulted in synergistic (CI < 1), additive (CI ~ 1), and predominantly antagonistic (CI > 1) effects, respectively. In imatinib-resistant K562-R and LAMA84-R cells, all applied drug combinations were synergistic (CI < 1) at higher growth inhibition levels. CONCLUSIONS: Our in vitro data warrant further investigation and may provide the basis for nilotinib-supplemented conditioning regimens for allo-SCT in advanced-phase CML.

Efficient gene transfer with pseudotyped recombinant adeno-associated viral vectors into human chronic myelogenous leukemia cells.

Gene transfer into chronic myelogenous leukemia (CML) cells may become of relevance for overcoming therapy resistance. Single-stranded pseudotyped adeno-associated viruses of serotypes 2/1 to 2/6 (ssAAV2/1-ssAAV2/6) were screened on human CML cell lines and primary cells to determine gene transfer efficiency. Additionally, double-stranded self-complementary vectors (dsAAVs) were used to determine possible second-strand synthesis limitations. On human CML cell lines, ssAAV2/2 and ssAAV2/6 were most efficient. On primary cells, ssAAV2/6 proved significantly more efficient (4.1 ± 2.5% GFP(+) cells, p = 0.011) than the other vectors (<1%). The transduction efficiency could be significantly increased (45.5 ± 13.4%) by using dsAAV2/6 vectors (p < 0.001 vs. ssAAV2/6). In these settings, our data suggest conversion of single- to double-stranded DNA and cell binding/entry as rate-limiting steps. Furthermore, gene transfer was observed in both late and earlier CML (progenitor) populations. For the first time, efficient AAV gene transfer into human CML cells could be shown, with the potential for future clinical application.

Synergistic activity of nilotinib and established chemotherapeutic drugs in imatinib-sensitive and -resistant BCR-ABL-positive cells.

We investigated various combination treatment regimens employing nilotinib with established chemotherapeutic agents (daunorubicin, mitoxantrone, etoposide and cytarabine) in imatinib-sensitive and -resistant BCR-ABL-positive cells. Mitoxantrone or cytarabine showed synergism (CI < 1) in combination with nilotinib in imatinib-sensitive LAMA84 cells, whereas in imatinib-resistant LAMA84-R cells synergistic effects could be assessed for daunorubicin, mitoxantrone and etoposide when combined with nilotinib. In both imatinib-sensitive and -resistant K562 cells daunorubicin, mitoxantrone and etoposide demonstrated synergism in combination with nilotinib. Moreover, both daunorubicin and mitoxantrone led to synergistic antiproliferative effects when combined with nilotinib in imatinib-resistant Ba/F3 cells carrying point mutations in the ABL TK domain (E255K, E255V and T315I). Annexin V/propidium iodide staining revealed a significant enhancement of nilotinib-induced apoptosis in imatinib-resistant Ba/F3T315I and LAMA84-R cells upon combination with daunorubicin and mitoxantrone, respectively. Our results demonstrate the efficacy of combination treatment regimens employing nilotinib and established chemotherapeutic agents in improving antileukemic effects in imatinib-sensitive and imatinib-resistant cells. This may be the foundation for further study on the potential of the applied combinations in a clinical setting.

Imatinib combined with mitoxantrone/etoposide and cytarabine is an effective induction therapy for patients with chronic myeloid leukemia in myeloid blast crisis.

BACKGROUND: Despite advances in drug therapy and allogeneic stem cell transplantation (allo-SCT), the prognosis of patients with chronic myeloid leukemia (CML) in blast crisis remains poor. Imatinib has demonstrated synergistic effects in vitro with mitoxantrone, etoposide, and cytarabine. METHODS: A Phase I/II trial was performed in patients with CML myeloid blast crisis. Patients were treated with imatinib + mitoxantrone/etoposide in four cohorts: mitoxantrone 10 mg/m(2)/day and etoposide 100 mg/m(2)/day for 2 or 3 consecutive days and imatinib 600 mg/day from Day 15 (cohorts 1 and 2) or from Day 1 (cohorts 3 and 4). After hematologic reconstitution after the cytopenic phase, cytarabine was given at a dose of 10 mg/m(2)/day in addition to imatinib as maintenance treatment. RESULTS: A total of 16 patients were available for analysis, median age 59 years (range, 37-74). All patients who received more intensive induction treatment (cohorts 3 and 4, n = 7) achieved a hematologic response (HR). In contrast, HR was achieved in only 6 of 9 patients treated in cohorts 1 and 2. The induction treatment was well tolerated. Six patients who achieved HR received an allo-SCT with myeloablative conditioning. The median survival in the transplant group was 16.2 months vs 4.7 months in the group with conventional treatment only (P = .067). CONCLUSIONS: The combination of mitoxantrone/etoposide and imatinib is well tolerated, with mild nonhematologic toxicity even in older patients. Eligible patients benefit from allo-SCT after response to the induction treatment.

Development and optimization of a real-time quantitative PCR-based method for the titration of AAV-2 vector stocks.

Despite the clinical application of adeno-associated virus (AAV) gene therapy, the titration of viral stocks has not yet been standardized. This complicates the comparison of viral stocks between laboratories. Functional titering of AAV is time-consuming, requires the manipulation of hazardous material, and often has a high degree of variability. We established an optimized real-time quantitative polymerase chain reaction (RQ-PCR) titration assay to determine viral titers and compared it with a functional green fluorescent protein (GFP)-based titration method. With a combination of improved lysis procedures and RQ-PCR protocols we could decrease the intraexperimental coefficient of variation (CV) from 0.24 +/- 0.03 to 0.042 +/- 0.004 and the interexperimental CV from 0.34 +/- 0.06 to 0.093 +/- 0.028 following functional and RQPCR-based titration, respectively. This low variability conforms to even the strictest quality standards required, for example, in clinical laboratories. The highly standardized titration by RQPCR described here will be especially advantageous for groups working on AAV-based gene therapy in a good manufacturing practice setting.

Clonal analysis of individual marrow-repopulating cells after experimental peripheral blood progenitor cell transplantation.

Methods to analyze the clonality of an adverse event in preclinical or clinical retroviral stem cell gene therapy protocols are needed. We analyzed the progeny of retrovirally transduced human peripheral blood progenitor cells (PBPCs) after transplantation and engraftment in immune-deficient mice. The integration site of the provirus serves as a unique tag of the individual transduced PBPC. A plasmid library of junctions between proviral and human genomic DNA was generated. We were able to detect individual transduced cell clones that amounted to 0.14%-0.0001% of chimeric bone marrow cells. This is the first report in which the contribution of individual marrow-repopulating cells to human hematopoiesis is directly quantified.