bluuebunny/crossref_metadata_2025 huggingface dataset:
Reproducibility and Robustness of the Xcellerate III Process for the GMP Manufacture of Xcellerated T Cells™ for Infusion into CLL Patients.
Authors: Hami Lisa , Green Cherie , Miller Katharine , Craig Stewart
Published: October 2019

Autologous peripheral blood mononuclear cells (PBMC) cryopreserved from a leukapheresis collection comprise the starting cellular source for the Wave® Bioreactor-based Xcellerate III Process [Hami et al, Bioprocessing Journal2003: 2; 23–32] used for the GMP manufacture of Xcellerated T Cells. For an ongoing clinical trial, n=13 patients have been infused with products manufactured from PBMC cryopreserved and stored in the vapor phase of liquid nitrogen (LN2) for 2–9 days before use in the Xcellerate III Process. This clinical protocol was recently amended to allow patients to receive a 2nd infusion of Xcellerated T Cells. To date, 2nd products have been manufactured for 11 of the CLL patients using the original PBMC that had been stored cryopreserved for up to 7 months from collection. Comparison of the processes for the manufacture of 1st (n=13) and 2nd (n=11) infusion products shows:

• No significant difference in the in-process T cell activation as determined by increase in cell size, up-regulation of CD25 & up-regulation of CD154 expression (refer to Figure 1).

Figure Figure

• The total cell yield for 2nd infusion products is within one (1) standard deviation of the average for the manufacture of the 1st infusion product (refer to Table 1).

• No significant difference in cell viability, CD3+ purity, or CD4:CD8 ratio for the final Xcellerated T Cells product (refer to Table 1).

Table 1. Final Product Characteristics

Final Product (Day 13) Average±S.D. p value 1st Infusion (n=13) 2nd Infusion (n=11) Total Cell Yield (x109) 0.01 137 ± 35 104 ± 17 Cell Viability (%) 0.15 93.5 ± 3.4 91.6 ± 2.4 CD3+ Purity (%) <0.001 98.4 ± 1.1 99.0 ± 0 CD4:CD8 Ratio 0.32 8.5 ± 7.7 5.7 ± 4.5

These data demonstrate high reproducibility and robustness of the Xcellerate III Process when using PBMC from the same leukapheresis collection in sequential processing runs. In addition, these data demonstrate that cryopreserved PBMC can be stored for many months prior to their use as the starting material in the Xcellerate III Process. Xcyte™, Xcyte Therapies™, Xcellerate™, Xcellerated T Cells™ and the circle logo are trademarks of Xcyte Therapies, Inc.
Mobilization of Peripheral Blood Progenitor Cells (PBPC) with G-CSF and GM-CSF in Autologous and Allogeneic Pediatric Transplants.
Authors: Echevarria Maria E. , Gonzalez Javier , Barrios Nilka J.
Published: October 2019

BACKGROUND: Chemotherapy and/or use of single or multiple growth factors have simplified the mobilization and collection of PBPC for autologous and allogeneic transplantation following high-dose chemotherapy. Nevertheless, the best method for PBPC mobilization continues uncertain. In children, mobilizations for CD34+ cells for autologous or allogeneic PBPC has been almost exclusively done with G-CSF. We conducted a phase III study to evaluate the results achieved using the combination of G-CSF + GM-CSF mobilizing CD34+ cells in children undergoing autologous or allogeneic PBPC transplantation.

METHOD: A total of 34 pediatric patients participated in the study. Seven healthy donors and 27 pediatric patients with different types of cancer participated in the mobilization study. They included patient with acute lymphoblastic leukemia, acute myelogenous leukemia, advanced stage neuroblastoma, Hodgkin’s lymphoma and rhabdomyosarcoma. Mobilization with G-CSF and GM-CSF (10μg/Kg/day each) was administered for 5 consecutive days in all patients, followed by PBPC harvesting until a threshold of 2.0 x 10^6/Kg CD34+ cells was obtained. A total of 19 PBPC transplants were performed (15 autologous and 5 allogeneic). Neutrophil engraftment was defined as ANC> 500/mm³ and, platelet engraftment was defined as a platelet count > 30,000/mm³ (transfusion independent) for more than 48 hours.

RESULTS: Table 1. The engraftment kinetics for neutrophils and platelets were 9.87 and 12.6 days respectively for the autologous transplants, and 9.4 and 12.2 days for the allogeneic. The mean number of days to achieve a higher or equal to 2 x 10^6 count of CD34+ cells in cancer patients was 2 days and 1 day for healthy donors. Side effects during mobilizations were mild and included nausea or vomiting, and tingling sensations in one individual. No secondary side effects like bone pain, headaches, or flu-like symptoms were experienced or documented. No infections were documented in the transplanted population, both autologous and allogeneic. Acute Graft versus Host Disease (GVHD) was documented in 2 patients that underwent an allogeneic PBPC transplants.

CONCLUSIONS: This study suggest that the combination of G-CSF + GM-CSF (10μ/Kg/day x 5 days) is well tolerated. It seems to be cost effective and adequate to mobilize more than average numbers of PBPC in a shorter period of time (from 1–3 apheresis) to perform allogeneic/autologous hematopoietic transplants in the pediatric population. It furnished a clinical reduction in the degree and duration of severe neutropenia in both allogeneic and autologous transplants as compared with other reports, without added increase incidence of GVHD.

Mobilizations

24 hrs 48 hrs 72 hrs 96 hrs Mean #CD34+ x 10^6/Kg (cancer patients) 3.3 2.56 1.43 0.82 Mean #CD34+ x 10^6/Kg (healthy donors) 6.7 5.1 12.2
Etoposide Containing Preparative Regimen for Allogeneic Hematopoietic Stem Cell Transplantation in Patients with Acute Lymphoblastic Leukemia.
Authors: Saito Takeshi , Asai Osamu , Dobashi Nobuaki , Yano Shingo , Ichiba Tamotsu , Osawa Hiroshi , Takei Yutaka , Sugiyama Katuki , Okawa Yutaka , Takahara Sinobu , Yamaguchi Yuko , Minami Jiro , Usui Noriko , Kobayashi Masayuki
Published: October 2019

[Purpose] Allogeneic hematopoietic stem cell transplantation (allo-HSCT) is one of the powerful therapeutic tools for patients with adult lymphoblastic leukemia (ALL) even though transplant-related toxicity and the recurrence of leukemia are still problematic. In order to improve outcome of patients with adult ALL who received allo-HSCT, we investigated the clinical efficacy and safety of etoposide containing conditioning regimen for allo-HSCT.

[Patients and Methods] Adult patients (including patients with 17, 18 and 19 years of age) with ALL were treated between February 1991 and October 2003 in our institute. When patients attained complete remission (CR) by induction therapies using JALSG ALL protocols or others, they received allo-HSCT [bone marrow (BMT), peripheral blood stem cell (PBSCT), and cord blood transplantation (CBT)]. Conditioning regimen was consisted with etoposide (60 mg/kg), cyclophosphamide (120 mg/kg), and fractionated total body irradiation (10 Gy) (CY/ETP/TBI). As clinically indicated, cytosine arabinoside (CY/ETP/TBI/AraC) or nimustine hydrochloride (CY/ETP/TBI/ACNU) was added to CY/ETP/TBI.

[Results] In the 23 patients [male/female: 15/8, median age was 30 years (range 17–50 years)], 19 patients received BMT (13 from related and 6 from un-related donor), 3 received PBSCT from related donor, and one was performed CBT. As the conditioning regimen, 16 patients received CY/ETP/TBI, 4 received CY/ETP/TBI/AraC, and 3 received CY/ETP/TBI/ACNU. Six out of 23 patients had Philadelphia chromosome positive ALL. Twenty-two patients received HLA serologically identical donor. Remission status at transplant included 16 patients in 1st CR, 5 in 2nd CR, and other 2 patients in non-CR. Grade II toxicity according to Bearman scale was observed in 52% (stomatitis), 22% (gastrointestinal), 9% (bladder), 4% (hepatic), and 4% (cardiac). Critical regimen-related toxicities and fatal veno-occlusive disease were not observed in any patients. The disease-free (DFS) and over-all survival (OS) rate of all patients was 51.1 % and 55.8 %.

[Discussion/Conclusion] These results indicated that an intensified etoposide containing preparative regimen is a feasible regimen and it has good efficacy for transplant recipients with ALL.
Defective Anti-Bacterial Carbohydrate Antibody Production after Unrelated HSC Transplantation.
Authors: Shah Ami J. , Wagner John E. , Kapoor Neena , Chan Raymond , Weinberg Kenneth I. , Parkman Robertson
Published: October 2019

Protective levels of anti-bacterial carbohydrate antibodies are necessary to protect HSC transplantation recipients from severe infections with encapsulated bacteria. Recipients of unrelated HSC transplants are at increased risk of severe infections regardless of their history of graft versus host disease (GVHD). Antibodies to a naturally occurring, prototypic bacterial carbohydrate antigen, polyribose phosphate (PRP), present in Hemophilus influenza type b and K100 stains of E. coli, were measured in the recipients of unrelated bone marrow (UBMT) and unrelated cord blood transplants (UCBT). UBMT and UCBT recipients, who were not on immunosuppressive therapy, were analyzed with follow-ups of 12 and 7 years, respectively. Of 20 UBMT recipients, only 3 had protective anti-PRP antibody levels. All 3 recipients with protective antibody levels were younger than 1 year old at the time of transplantation and did not have a history of GVHD. No recipients, who were older than 1 year old at the time of transplantation, had protective levels of anti-carbohydrate antibody. Of 16 UCBT recipients, 7 had protective antibody levels, including recipients as old as 53 at the time of UCBT. All unrelated recipients, who did not have protective levels of anti-carbohydrate antibodies, were able to produce protective levels of antibodies to a protein antigen, tetanus toxoid, after immunization. Therefore, the inability of the recipients of unrelated HSC to produce protective levels of anti-bacterial carbohydrate antibody is not part of a generalized antibody deficiency state, but is a specific defect. Since bacterial carbohydrate antigens are T lymphocyte-dependent antigens in humans, the results indicate that thymic and T lymphocyte function are greater in UCBT recipients than in UBMT recipients (p=.008 for protective anti-PRP antibody levels in recipients older than age 1). Defects in protective anti-bacterial carbohydrate antibody production are common in the recipients of HSC unrelated transplants regardless of the source of HSC. The recipients of unrelated HSC transplants, especially UBMT, should be tested for their capacity to produce anti-carbohydrate antibody, and, if deficiencies are detected, appropriate prophylactic measures undertaken to reduce the likelihood of severe bacterial infections.
Management of Febrile Neutropenia: Single Agent or Combination Therapy?.
Authors: Caldas Joana , Fernandes Teresa , Monteiro Alexandra , Carande Luisa , Silva Madalena , Cruz Ernesto , Sousa Aida B.
Published: October 2019

The best strategy for the empirical treatment of fever in profoundly neutropenic patients (pts) remains unclear. We retrospectively compared the efficacy and safety of 2 antibacterial regimens consecutively used in our center for febrile neutropenia (FN) in pts undergoing intensive chemotherapy for acute leukemia or autologous stem cell transplantation. From 1/99 to 6/01, 374 episodes of FN in 179 pts were treated with piperacillin-tazobactam plus amikacin (PTA, 4.5 g q6h and 20 mg/kg q24h respectively), then from 7/01 to 12/03, 428 episodes in 223 pts were treated with cefepime (CEF, 2g q8h). Any modification of the initial regimen was evaluated as a failure. Pts were nursed in HEPA-filtered rooms and received prophylactic aciclovir (and ciprofloxacin until 12/99) with no antifungal primary prophylaxis. There were no significant differences between the PTA and CEF episodes regarding median age of the pts, median days with neutrophils <500/μl or presence of an intravenous central catheter. The proportion of documented bacteremias was similar (33/30%) with a Gram negative predominance in both periods (60/66%), mainly due to E. coli (61/64%) and P. aeruginosa (14/14%). As for Gram positive bacteremias, there was an increase in S. aureus (18% in PTA vs 27% in CEF) and a marked decrease in S. viridans (24/4%). Resistance of Gram negative bacteria to the protocol drugs remained low (7% to PT and 4% to A in the first period, 5% to CEF in the second); resistance of coagulase negative staphylococci to ciprofloxacin dropped from 33 to 15%. Success rates were 35% for PTA and 32% for CEF. The reasons for modification of the initial empirical regimen were similar in the 2 periods, the most frequent being persistent fever (56/59%) followed by progression of infection (20/18%), relapsing fever (19/14%), withdrawal due to toxicity (3/6%) and resistant pathogen (1/3%). The addition of an antifungal was the most usual modification, occuring in 44% of PTA and 39% of CEF episodes. Vancomycin (for which a restriction policy had been introduced in 1995 after an outbreak of vancomycin-resistant enterococci) was added in 17 and 15% of the episodes. Amikacin was added in 38% of the CEF episodes. The occurence of further infections did not differ between the 2 periods (26 and 29%) nor did the mortality due to infection (7.7 and 6%). The proportion of severe adverse events (CTC-NCI grades 3/4) was low in both periods (4 and 1%). Cutaneous allergic reactions were less frequent with PTA than with CEF (5 vs 9%). We conclude that monotherapy with CEF was as effective as the combination of PTA for empirical treatment of FN. Both regimens resulted in a low infectious mortality and a very low emergence of resistance, and both were well tolerated.
MRI-Detectable Focal Lesions (FL) in Multiple Myeloma (MM) at Relapse Frequently Involve Novel Sites Not Involved at Diagnosis.
Authors: Walker Ronald , Jones-Jackson L. , Rasmussen Eric , Miceli M. , Anaissie Elias , Epstein Joshua , Tricot Guido , Shaughnessy John , Barlogie Bart
Published: October 2019

Macrofocal lesions (FL) in the medullary space are a hallmark of mm readily detected by MRI scans, both at diagnosis and at relapse. In this study we evaluated, among 25 patients enrolled in TT II, with FL at relapse and a preceding clinical CR (by M protein and marrow examinations) the relationship to presence and location of FL at diagnosis. On relapse, 17/25 (68%) had fewer FL, 5/25 (20%) had the same number of FL, 3/25 (12%) had more FL than on baseline examination. There was a general trend for the larger FL at baseline to still be present on relapse (Table 1). Despite the trend to fewer focal lesions on relapse compared to baseline, 11/25 (44%) of patients presented with FL in new sites that were not present on baseline. Finally, in relapsing patients, 4/25 (16%) presented with new sites of extramedullary tumor (EMD). These data establish that although there are usually fewer MRI-defined FL on relapse than on baseline examination, the vast majority of FL on relapse represent new areas of macrofocal disease that were not present at diagnosis.

MRI FL Persisting from Baseline to Relapse (n=25)

Size (cm) Baseline FL Present upon Relapse % < 0.5 1/2 50 0.5–1cm 8/10 80 1–2 cm 13/18 72 > 2 cm 10/16 63 All Sizes 26/36 72
A Matched Pair Retrospective Comparison of Adult Unrelated and Sibling Donor Allogeneic Transplantation for AML.
Authors: Moore John J. , Bradstock Ken B. , Dodds Anthony , Szer Jeff , Smith Ian Nivison , Goh Kim , Ma David
Published: October 2019

Aim: Recent studies have shown equivalent survival outcomes for unrelated donor transplants for CML compared to siblings. This study compares outcomes for a set of unrelated donor transplants for AML and a strictly selected matching set of sibling donor transplants.

Method: A set of 100 adult first 6/6 compatible unrelated donor transplant recipients with AML were selected from the ABMTRR database. A set of 100 adult first 6/6 compatible sibling donor controls were selected, matched with the subjects on disease stage, sex and age. Additional data was collected from participating centres on conditioning, prophylaxes, GVHD and other outcome factors.

Results: A total of 162 forms were returned: 22 for good risk (CR1 - 15 siblings, 7 unrelated) and 140 for poor risk (past CR1 - 77 siblings, 63 unrelated). A Kaplan-Meier comparison of post transplant disease free survival between good risk sibling and unrelated donor recipients was not statistically significant (p=0.2). However the 5-year DFS of poor risk unrelated recipients was significantly worse than sibling recipients (sibling 29.5%, unrelated 14.1%, p=0.01). In a multivariate Cox regression model, the independent risk factors for DFS among all patients were unrelated donor (p=0.003), disease stage beyond CR1 (p=0.04), recipient male (p=0.04), aged 40 and over (p=0.04) and CMV positive (p=0.03).

Conclusion: Adult unrelated donor allograft recipients in poor risk AML have poorer DFS probability than sibling recipients. However in good risk AML allograft recipients, donor type is not a significant DFS risk factor. The ABMTRR is an important national data resource providing a large and readily accessible sample frame for retrospective studies on specific transplant indications.
Peg-Filgrastim after Autologous Peripheral Blood Stem Cell Transplantation in Hematological Malignancies.
Authors: Musto Pellegrino , Rosario Scalzulli Potito , Melillo Lorella , Nobile Michele , Dell’Olio Matteo , La Sala Antonio , Mantuano Saverio , Bodenizza Carlo , Falcone Antonietta , Sanpaolo Grazia , Michele Carella Angelo , Greco Michele Mario , Beltrami Germana , Cascavilla Nicola
Published: October 2019

ASCO guidelines recommend the use of granulocyte-colony stimulating factors (G-CSF) as adjuvant agents after autologous peripheral blood stem cell transplantation (PBSCT), for shortening the period of severe neutropenia and reducing the related risk of life-threatening infections. The recently available pegylated formulation of G-CSF (peg-filgrastim) could have some potential advantages in phase of recovery after PBSCT, such as self-regulation mechanisms, particularly useful when neutropenia duration is unforeseeable, possible reduction/standardization of growth factor costs and possibility of more manageable outpatient treatments. We investigated the effects of a single s.c. injection of peg-filgrastim (6 mg) on day 3 in 11 patients (8 with multiple myeloma, MM; 2 with non-Hodgkin’s lymphomas, NHL; 1 with acute myeloid leukemia, AML) planned to receive autologous PBSCT. Mean age was 55 years (range 24–65). In 7 patients PBSCT was performed as consolidation in first complete or partial remission, while 4 patients received the procedure as salvage treatment for chemo-sensitive disease. Conditioning regimens were intermediate/high dose melphalan in MM, BEAM protocol in NHL and TBI plus cyclophosphamide in AML. For comparison, we selected a group of 11 historical controls matched for age, type and status of disease, conditioning regimen, and number of CD34+ stem cells infused, who had received standard daily s.c. administration of G-CSF (300 mcg/d) from day 5 to neutrophil recovery after autologous PBSCT. No adverse events occurred in patients treated with peg-filgrastim. Bone pain was the only side effect referred by one patient. There was no difference between the two groups in terms of median time to hemoglobin recovery, days of febrile neutropenia and number of red cell or platelet transfusions received. However, the median times to neutrophil > 0.5 x 109/L (10 days, range 9–13 vs 12 days , range 11–14, p < 0.000) and platelet > 20 x 109/L (10 days, range 8–15 vs 13 days, range 12–20, p < 0.003) were significantly shorter in peg-filgrastim group. The median number of G-CSF injections in controls was 12 (range 9–20), so that the mean cost of this drug for single patient was 1,764 euros, as compared to 910 euros for patients receiving peg-filgrastim. We conclude that peg-filgrastim and G-CSF have comparable safety and efficacy profiles in patients affected by hematological malignancies who undergo autologous PBSCT. In this specific setting of patients, peg-filgrastim could be cost-effective. Whether peg-filgrastim may be also superior in reducing the periods of neutropenia and thrombocytopenia, requires to be confirmed and further investigated in larger studies.
Follow-Up of Chronic Lymphocytic Leukemia (CLL) Patients Who Relapse after Autologous Stem Cell Transplantation (ASCT).
Authors: Trisolini Silvia M. , Mauro Francesca R. , Capria Saveria , Cimino Giuseppe , De Propris Maria S. , Proia Anna , Foà Robin , Meloni Giovanna
Published: October 2019

It is now clear that ASCT despite being a feasible and potentially beneficial therapeutic strategy in the treatment of CLL patients (pts), does not lead to cure. Whether, how and when to utilize this procedure in the course of the disease is still matter of debate. In order to evaluate whether or not pts relapsed after an ASCT could be retreated, also with intensive approaches, we evaluated the outcome of our CLL pts with recurrence of disease after an ASCT. Between 1995 and 2002, 30 CLL pts, median age 50 years (range 24–61), with advanced disease (Binet stage B or C), in clinical complete remission (CR) after fludarabine (FLU), were autografted at our institution using bone marrow (4 pts) or peripheral blood stem cells (26 pts); the BEAM (BCNU, etoposide, ara-c, melphalan) conditioning regimen was utilized in most pts (24). At the time of transplant, 17 pts had received one line of therapy, 8 two lines and 5 three or more lines, and the median interval from diagnosis to transplant was 41 months (range 8–131). Eighteen of the 30 pts (60%) showed a clinical relapse after a median time of 31.5 months (range 2–79) from transplant. All relapsed pts required treatment which was administered after a median interval from relapse of 4 months (range 1–38). Seventeen pts were evaluable for response, 1 pt being too early. Two of the 17 pts proved refractory to two lines of therapy (chlorambucil (CB), FLU plus cyclophosphamide (CY): 1 pt; CB, vincristine: 1 pt) and died 28 and 32 months from relapse, respectively. Fifteen of the 17 pts (88%) achieved a response. A partial response was obtained in 12 pts after first line therapy (CB: 6 pts; mabthera alone: 3 pts; FLU-CY and mabthera: 2 pts; PVABEC regimen: 1 pt). A CR was achieved after one line of therapy (FLU-CY and mabthera) in 1 pt and after a second line of therapy in 2 pts (mabthera, FAND plus allo: 1 pt; CB, FLU plus campath-1H: 1 pt). The median response duration was 16 months. At the present time, 11/18 relapsed patients are alive with a median follow-up of 37 months from relapse (range 18–63) with a projected probability of overall survival of 40% at 5 years from relapse. Seven patients have died because of disease progression after a median interval of 36 months from relapse (range 28–50). Based on our results, ASCT does not jeopardize the possibility of submitting CLL patients who relapse after such a procedure to further treatment, also intensive, which can induce good and lasting responses with a possibility of survival of 40% at 5 years from relapse.
Evaluation of Yttrium 90 (90Y) Ibritumomab Tiuxetan (Zevalin®) in Patients with Non-Hodgkin’s Lymphoma (NHL) Having Previously Received Autologous Stem Cell Transplant (ASCT).
Authors: Jacobs Samuel A. , McCook Barry , Torok Frank , Avril Norbert , Vidnovic Nick , Joyce Judy , Molina Arturo
Published: October 2019

Background: As the first radioimmunotherapeutic (RIT) agent approved for the treatment of relapsed or refractory B-cell NHL, yttrium 90 (90Y) ibritumomab tiuxetan (Zevalin®) has been shown to achieve durable responses in heavily pretreated NHL patients. In phase 1–2, the eligibility criteria for 90Y ibritumomab tiuxetan therapy excluded patients with hypocellular bone marrow (<15%), lymphomatous involvement >25%, or prior ASCT due to concern of depleting the bone marrow reserve. However, Kaminski et al previously demonstrated that a parallel RIT agent, 131I tositumomab, was safe and efficacious when administered after ASCT (Blood2000; 96:1259–66). We report our experience of using ibritumomab tiuxetan in NHL patients with a prior history of ASCT.

Methods: Patients with histologically confirmed relapsed or refractory B-cell NHL, ≥18 years, platelet counts >100,000/mm3, bone marrow cellularity >15%, and lymphomatous involvement <25% were eligible for treatment. In a series of 40 patients treated with ibritumomab tiuxetan at the UPMC Cancer Center, 6 patients with prior ASCT were treated. A standard course of ibritumomab tiuxetan was given, with the therapeutic dose of 90Y ibritumomab tiuxetan administered at 0.3 mCi/kg (the initial patient, and another with platelet count ≥150,000/mm3) or the standard dose of 0.4 mCi/kg 90Y in the 4 remaining cases (baseline platelet count >150,000/mm3). Pretreatment diagnostic PET/CT scans were taken for all patients and evaluated for areas of lymphomatous involvement. Follow-up scans were performed approximately 12 weeks after treatment to assess patient response. Maximum toxicities were monitored weekly over a 12-week period after therapy and classified according to CTCAE v. 3.0 toxicity criteria.

Results: Patients with a prior history of ASCT had received a minimum of 4 previous regimens (range, 4–7). Subjects presented with follicular NHL (n = 3), transformed NHL (n = 1), large cell lymphoma (n = 1), and mantle cell lymphoma (n = 1). Six patients were evaluable for toxicity and 5 patients were evaluable for response. Observed toxicities were consistent with those expected in this patient population, with 33% (2/6) and 17% (1/6) of patients experiencing grade 4 thrombocytopenia and grade 3 neutropenia, respectively. Episodes of bleeding or neutropenic fever were not observed. Of the 5 patients evaluable for response, 1 patient with follicular lymphoma had a complete response to ibritumomab tiuxetan as determined by FDG-PET imaging.

Conclusions: Ibritumomab tiuxetan therapy is feasible and safe in NHL patients who have previously received ASCT. Heavily pretreated patients can benefit from the administration of ibritumomab tiuxetan although additional data are needed to further characterize the degree of clinical response after ASCT.
Autologous Peripheral Blood Stem Cell Transplant for Relapsed or Refractory T-Cell Non-Hodgkin’s Lymphoma Results in Inferior Long Term Survival when Compared to Relapsed or Refractory B-Cell Non-Hodgkin’s Lymphomas.
Authors: Hsu Jack W. , Singh Priya , Goldberg Stuart L. , Pecora Andrew L. , Siegel David S. , Rowley Scott D.
Published: October 2019

Autologous transplant has emerged as the standard of care for the treatment of relapsed B-cell non-Hodgkin’s lymphomas (NHL). However, for the treatment of T-cell NHL, the role is not as clear. We report our experience with 24 patients with T-cell NHL who underwent autologous transplantation at time of first relapse. Lymphoma types were varied (peripheral T-cell = 8, lymphoblastic = 6, anaplastic large cell = 5, not otherwise specified = 5). All patients received CHOP as primary therapy, except one patient who received ProMACE/CytaBOM, one who received Stanford V, and two who received HyperCVAD. Patients had either relapsed disease (14 patients) or primary refractory disease (9 patients). One patient underwent autologous transplant as consolidation therapy. Mobilization regimens varied, consisting of dexamethasone, ifosphamide, etoposide, and cisplatin (DICE, 12 patients), dose intensive cyclophosphamide, etoposide, cisplatin (DICEP, 5 patients), cyclophosphamide/etoposide (3 patients), cyclophosphamide (2 patients), or other regimens (2 patients). Adequate collection of stem cells was seen in all but one case, which required bone marrow collection. The conditioning regimen used was BVAC (BCNU 600 mg/m2, etoposide 1600 mg/m2, cytarabine 24 g/m2, and cyclophosphamide 90 mg/kg) in 23 patients. One patient received melphalan conditioning. For the entire group, the overall survival at 3 years was 50%. This compares favorably with 336 relapsed B-cell NHL patients who underwent autologous transplant with the identical conditioning regimen (BVAC) in the same period of time (OS = 52%). At 5 years, the overall survival of the B-cell lymphoma group was stable at 49%. However, the overall survival for the T-cell NHL group declined to 30%. The primary cause of death post transplant was disease relapse. When divided among specific lymphoma subtypes, 3 of 8 patients with peripheral T-cell lymphoma, 4 of 5 patients with anaplastic large cell lymphoma, 3 of 6 patients with lymphoblastic lymphoma and 3 of 5 patients with other T-cell lymphoma subtypes remain alive. Although there are too few patients to draw any definite conclusions, it appears that autologous stem cell transplant for relapsed or refractory T-cell NHL results in similar 3-year overall survivals when compared to B-cell NHL using the same conditioning regimen. However, T-cell NHL appear to be associated with a higher late relapse rate which is reflected in the lower overall survivals at 5 years. Although there may be a benefit to autologous transplant in relapsed anaplastic large cell lymphomas, relapsed/refractory T-cell NHL had inferior long term overall survival compared to their B-cell counterpart transplanted with the same regimen. Novel approaches should be considered in the treatment of relapsed or refractory T-cell NHL.
Co-Epression of Two Transgenes in Lentiviral Vector Using Independent Transcriptional Units.
Authors: Schuening Friedrich G. , Zaboikin Michail M. , Srinivasakumar Narasimhachar , Zaboikina Tatiana N.
Published: October 2019

There are several gene therapy approaches, which require transfer and co-expression of two transgenes within one target cell. To this end, we have created and tested two-gene expression HIV-1 based vectors, which encode enhanced green fluorescent protein (EGFP) and P144K mutant of canine O6-methylguanine-DNA-methyltransferase (MGMT) transgenes under either the phosphoglycerate kinase gene promoter (PGKp), or the elongation factor 1 alpha promoter (EF1a_p). Eight different configurations of the two transgene expression cassettes were created and tested within the same lentiviral backbone (see Figure). Individual VSV-G pseudotyped vectors stocks were produced and used for infection of canine thyroid adenocarcinoma (CTAC) cells at low multiplicity of infection (MOI = 0.1) to ensure 1 copy of proviral vector per transduced cell. The cells were harvested one week later and an aliquot was assayed for EGFP expression by flow cytometry. Another portion was subjected to selection with O6-benzylguanine (BG, 40 m M for 18 hrs) and 1,3-bis(2-chloroethyl)-1-nitrosourea (BCNU, 100 m M for 2 hrs) and kept in culture for additional two weeks to eliminate cells expressing insufficient MGMT. The percentage of GFP positive cells, prior to selection with BG and BCNU, ranged between 0.1 % to 6.6% for the dual-transgene expression cassette encoding HIV-1 vectors. Following selection with BG and BCNU, the percentage of GFP positive cells increased for all vectors with the exception of vector #2 PGEM. Two of the vectors (#1 PMEG and #8 EGMP) demonstrated over 80% GFP positivity after selection. The results of flow cytometry after selection were corroborated by fluorescence microscopy of individual BCNU-resistant colonies. GFP expression was readily detected in drug-resistant colonies transduced with vectors #1 PMEG or #8 EGMP. Weaker GFP expression was detected in drug resistant cells transduced with vectors #3 PMGE, #5 EMPG or #6 EGPM. No significant GFP expression was observed in drug-resistant colonies transduced with vectors #2 PGEM, #4 PGME or #7 EMGP. Drug resistance to BCNU (IC50 values) provided by each of the vectors, was also determined. The data showed that the IC50 values for #1 PMEG and #8 EGMP vectors were 2.4-fold and 4.4-fold, respectively, higher than for mock transduced control cells. The above results indicate that coordinated co-expression of two transgenes using independent expression cassettes is promoter, position and orientation dependent. The data also indicate that two potentially useful vectors (#1 PMEG and #8 EGMP) have been identified for evaluation, ex vivo and in vivo, in the canine model for co-expression of two transgenes.
Targeting Hematopoietic Cells Using a Blimp-1 Promoter Helper Dependent Adenoviruses.
Authors: Shi Chang-Xin , Wen Xiao-Yan , Wang Ding-Yan , Gao Bo , Sleep Gillian , Zhuang Li-Hua , Li Zhi-Hua , Hitt Mary M. , Stewart A. Keith
Published: October 2019

B lymphocyte-induced maturation protein-1 (Blimp-1) plays a key role in the maturation of B lymphocytes into end-stage, antibody-screting plasma cells. However its promoter region has not been well characterized. As an initiative aimed at identifying plasma cell specific promoters for use in gene therapy of myeloma, we studied the function of the blimp-1 promoter in a helper-dependent adenovirus (HDAd) vector system. The advantages of employing HDAd include: 1) High capacity encompassing transgene (upto 36 kb); 2). Higher and longer transgene expression; 3). Lower cytotoxicity in vivo; 4). Improved specificity for tissue-specific promoters. Blimp-1 promoter fragments of 2.6, 4.7 and 25 kb driving the luciferase gene were cloned into HDAd precursor plasmid and then rescued into HDAd vectors. A panel of B lymphoma and myeloma cell lines were infected with these HDAd vectors and a positive control vector HDMCMV (an HDAd vector expressing luciferase under the control of MCMV promoter) at an MOI of 2,000 particles/cell. Two days after infection, very high luciferase activity was demonstrated in human myeloma cell lines (KMS11, U266, MM1, and MY5 cells), but low or no activity was seen in human B lymphoma cell lines (Namalwa and Raji). In fibroblasts, the Blimp-1 derived promoter activity appeared to be high in COS7 cells but 10 times lower in NIH3T3 cells. All three differently sized promoter fragments of Blimp-1 demonstrated similar activity. Next we further tested these three Blimp1/Luc HDAd vectors in mice using HDMCMV as a positive control. Six week old FVB mice were intravenously injected with 1X10(e11) particles per mouse. Different tissues were assayed for luciferase activity three days after injection. The control HDMCMV virus directed the highest luciferase activity in the liver because of the tropism of adenovirus. However, the Blimp-1/luciferase viruses directed highest luciferase expression in the spleen and bone marrow with lower level relative expression in the liver and trace amount or no expression in other tissues examined. After normalization, the transciptional activity of the Blimp-1 promoter is ~80 fold higher than the CMV promoter in spleen tissue. All three Blimp-1 promoters demonstrated similar activity, indicating that the 2.6 kb Blimp promoter contains the regulation element for spleen and bone marrow-specific gene expression. FACS analysis of spleen cells co-stained with luciferase antibody revealed luciferase expression in both B220+ve B and B220-ve non-B cells. Taking together, we conclude that 2.6kb blimp-1 promoter sequence is enough to direct gene expression to hematopoietic cells but is not B cell specific.
Gene Transfer in Hematopoietic Progenitor Cells Mediated by an SV40 Based Episomal Vector Carrying A S/MAR Element.
Authors: Papapetrou Eirini , Ziros Panos , Mitcheva Ilina , Athanassiadou Aglaia , Zoumbos Nicholas
Published: October 2019

Background/Aims: Replicating episomal vectors present a potential alternative to currently used oncoretroviral vectors for gene transfer in hematopoietic progenitor/stem cells. Their main advantage is that they can persist in the recipient nucleus as independent units, without integrating into the host’s genome, eliminating thus the risk of insertional mutagenesis. In the present study we explored the capacity of a recently developed SV40-based episomal vector, pEPI-eGFP, to stably transfect hematopoietic progenitor cell lines and primary cells, in order to evaluate its potential for therapeutic applications. pEPI-eGFP contains the enhanced green fluorescent protein (eGFP) cDNA and a Scaffold/Matrix Attachment Region (S/MAR) and it does not code for any proteins of viral origin. These unique properties qualify pEPI-eGFP as an attractive vehicle for gene therapy applications.

Results: The vector was maintained as a stable episome in K562 cells for at least 100 generations and supported long-term EGFP expression, even in cells cultured in non-selective medium. Methylation-dependent cleavage assays demonstrated the vector’s ability to self-replicate in K562 cells and MEL cells, whereas its episomal status was confirmed by Southern blotting and plasmid rescue in E. coli. The vector was also maintained in primary human fibroblasts for at least 30 passages with and without selection pressure.

Transfection of CD34+ cells from umbilical cord blood with pEPI-eGFP was feasible by electroporation with an efficiency of up to 30%, as estimated by flow cytometric evaluation of eGFP expression 24–48 h post-transfection. Cytokine prestimulation of CD34+ cells did not enhance transfection efficiency, whereas transfection of post-mitotic cells, such as dendritic cells was also feasible. This suggests that efficient transfection with the vector does not require cell cycling. PCR with eGFP-specific primers in single CFU colonies derived from CD34+/eGFP+/PI− FACS-sorted cells demonstrated the presence of the vector in ~20% of the colonies.

Conclusion: Our results demonstrate that pEPI-2 exhibits a considerable potential as a gene transfer vector for hematopoietic cells, as it confers long-term transgene expression in hematopoietic progenitor cell lines as a stable episome and can efficiently transfect unstimulated CD34+ cells. Further studies, both in vitro and in vivo, are required to assess the long-term maintenance in hematopoietic progenitor/stem cells and their progeny as well as other features of the vector, in order to evaluate its overall efficacy and possibly improve its performance.
Lentiviral Delivery of Suicide Gene and Selection Molecules to Non-Dividng T Cells.
Authors: Qasim Waseem , Chatziandreou Ilenia , Thrasher Adrian , Gaspar Hubert
Published: October 2019

T cell suicide gene therapy is designed to permit the use of donor T cells for their powerful anti-leukaemic and anti-viral effects following haematopoietic stem cell transplantation. In the event of graft versus host disease (GVHD) such cells can be eliminated through the administration of suicide-gene dependent prodrugs. Though the feasibility of the strategy has been demonstrated in clinical trials, there has been a lower incidence of GVHD than anticipated and evidence of impaired anti-viral immunity. Current vectors under clinical investigation are based on retroviral delivery systems and require T cells to be actively dividing at the time of vector exposure to for successful transduction. The process of ex-vivo activation to induce mitogenesis has been shown to impair subsequent functional potential, and even under optimized conditions using combinations of anti-CD3 and anti-CD28 antibodies, T cells become prone to exhaustion. It has been previously reported that lentiviral vectors permit T cell transduction without the need for mitogenic stimulation if used in combination with certain cytokines such as Interleukin-2 (IL-2) or Interleukin-7 (IL-7). Thus we have adapted a self-inactivating lentiviral vector system based on HIV-1 and encoding strong promoter elements derived from the long terminal repeat (LTR) regions of Spleen Focus forming virus (SFFV) for the delivery of suicide genes to T cells. The Woodchuck hepatitis virus post-transcriptional regulatory element and the central polypurine tract element of HIV-1 have been incorporated to enhance transduction efficiency and increase transgene expression. T cells cultured in the presence of either IL-2 or IL-7 alone, or in combination could be transduced at between 20–30% efficiency following a single round of exposure to virus pseudotyped with the vesicular stomatitis virus envelope or the feline endogenous leukaemia virus envelope. By the end of the procedure there were minimal changes in T cell surface phenotype, preservation of niave/memory subsets and retention of virus specific populations as quantified by tetramer staining of Cytomegalovirus (CMV) specific T cells. Functional analysis indicated preservation of proliferative responses to autologous dendritic cells pulsed with CMV antigen. In addition, strong responses in mixed lymphocyte culture indicated intact allo-reactive responses. Cells transduced to express a fusion construct encoding a variant herpes simplex thymidine kinase fused to the truncated CD34 selection marker could be enriched to >95% purity by a single round of anti-CD34 magnetic bead selection. The functional integrity of the suicide gene was confirmed by elimination of enriched cells following exposure to the prodrugs Gancilcovir and Aciclovir. In conclusion, non-dividing T cells transduced with suicide gene/selection transgenes using lentiviral constructs, retain phenotypic characteristics and functional responsiveness.
IL-2/B7.1 Fusagene Transduction of AML Blasts by a Self-Inactivating Lentiviral Vector Stimulates T Cell Responses in Vitro: A Strategy To Generate Whole Cell Vaccines for AML.
Authors: Chan Lucas , Hardwick Nicola , Darling Dave , Galea-Lauri Joanna , Gäken Joop , Devereux Stephen , Kemeny Mike , Mufti Ghulam , Farzaneh Farzin
Published: October 2019

Despite recent treatment advances prognosis for the majority of AML patients remains bleak. AML patients over 60 years of age treated with intensive chemotherapy have an estimated 2-year survival of 20% and only 10% at 4–5 years. This is attributed to unfavourable cytogenetics, preceding myelodysplastic syndrome and limited abilities to tolerate intensive chemotherapy. We and others have previously demonstrated that tumour cells engineered to express B7.1 and IL-2 can stimulate T cell and NK cell responses against the unmodified tumour cells and prolong the survival of vaccinated animals in mouse models of leukaemia. However, development of whole cell vaccines for AML have been severely hampered by the fact that primary AML cells are poorly infectable with RV, AdV, AAV and HSV. Therefore we have constructed self-inactivating lentiviral vectors incorporating enhancer elements and a myeloid efficient promoter to express B7.1 and IL-2 in a monocistronic “fusagene” configuration as a single fusion protein. This polyprotein is post-synthetically cleaved to generate biologically active membrane anchored B7.1 and secreted IL-2. This provides transduction efficiencies of between 40%–99% for B7.1 and 2-15ng/106/24hrs of IL-2 secretion in both established and primary AML cells, following a single round of infection at an MOI of 10. The combined expression of IL-2 and B7.1 in AML blasts enables increased in vitro stimulation of allogeneic T cells and autologous T cells obtained from both non-remission and remission patients (post-BMT or non-BMT recipients). In addition, IL-2/B7.1 AML cells stimulated an increase in lymphocyte numbers, expansion of CD8+ cells and increased expression of T cell activation markers. Furthermore, the stimulated lymphocytes secrete greater levels of Th1 cytokines and show evidence of specificity, as indicated by their increased proliferation in the presence of unmodified AML compared to remission bone marrow cells. This data supports the notion that IL-2/B7.1 whole cell vaccination could stimulate therapeutically useful immune responses in AML patients.
Long-Term Follow-Up of 98 Cases of Recombinant Human Erythropoietin (epoetin) Induced Pure Red Cell Aplasia (PRCA): Outcomes and Treatment Data from the Research on Adverse Drug Reactions and Reports (RADAR) Program.
Authors: Carson Kenneth R. , Klinge Stephen A. , Belknap Steven M. , McKoy June M. , Raisch Dennis W. , Tallman Martin S. , Kuzel Timothy M. , Evans Andrew M. , Kim Benjamin , Yarnold Paul R. , Lyons Elizabeth A. , Bennett Charles L.
Published: October 2019

Background: With the recent reporting of 191 cases of epoetin induced PRCA (Bennett CL, NEJM 2004), information is needed about the long-term follow-up of individuals with this diagnosis. While 47 cases from France, Germany, and England have been reported recently (Verhelst D, Lancet 2004), these data are limited by short follow-up and absence of information from other regions.

Methods: RADAR investigators identified cases of epoetin-associated PRCA from the Food and Drug Administration MEDWatch database and proprietary reports from both the manufacturers epoetin alpha (Epogen from AMGEN, and Eprex from Johnson and Johnson) and epoetin beta (Neorecormon from Roche). Patients exposed to more than one epoetin form were excluded. PRCA recovery was operationally defined as transfusion independence, clearance of anti-erythropoietin antibody, and/or normalization of reticulocyte count.

Results: See table.

Conclusions: The overall recovery rate of 82% following transplantation versus 40% following immunosuppression suggests that transplantation might be the optimal therapy for epoetin-associated PRCA. Almost half of the patients rechallenged with epoetin developed recurrent PRCA, raising concern over the benefits of rechallenge.

Eprex (N=82) Neorecormon (N=11) Epogen (N=5) Origin: US 60% 64% 100% Europe 60% 64% – Pacific Rim/Asia 21% 36% – Canada 19% – – Subcutaneous delivery 99% 100% 60% Recovery with Immunosuppression 37% 73% 0% Recovery with renal Transplant 11% – – No recovery with renal transplant 1% – 20% No Recovery 41% 18% 40% Unknown Recovery 10% 9% 40% Male gender 68% 73% 40% Rechallenged with epoetin N=12 – – Response rate 50% Median age 68 years 47 years 51 years
Establishment of a Retroviral Mouse Model for inv(16)-Mediated Acute Myeloid Leukemia Suggests That the p14ARF Tumor Suppressor Is a Transcriptional Target for Repression by the inv(16) Fusion Protein.
Authors: Moreno Isabel , Pan Ling , Keates Jennifer , Kim Hyung , Klug Christopher , Hiebert Scott
Published: October 2019

The inv(16) is one of the most frequent chromosomal translocations associated with acute myeloid leukemia. The chimeric gene encodes a fusion protein consisting of most of the RUNX1 co-factor core binding factor beta (CBFβ) fused to a smooth muscle myosin heavy chain, SMMHC. Given that RUNX1 and RUNX1-ETO regulate the p14ARF promoter, we tested whether the inv(16) fusion protein (IFP) can also repress p14ARF expression. In reporter assays, the IFP cooperated with RUNX1 to repress the p14ARF promoter. To investigate whether the IFP can regulate endogenous ARF expression, we triggered the oncogene checkpoint and the expression of p19ARF (the murine homologue) by expressing c-Myc in primary myeloid progenitor cells. After induction of c-Myc activity, p19ARF levels increase 20–50 fold; however, the co-expression of the IFP and c-Myc effectively blunted the induction of p19ARF. Next, we sought to take advantage of this information to establish a murine model of the inv(16). We reasoned that the IFP provides a growth advantage and perhaps allows mutations to accumulate by repressing p19ARF. If so, then removing p19ARF during the rapid growth of hematopoietic stem cells and progenitors during embryogenesis might allow the formation of the same mutations that would occur after expression of the IFP, but accelerate leukemogenesis upon IFP expression. Therefore, we used p19ARF-null mice as bone marrow donors and used recombinant retroviruses to express the IFP together with a green fluorescent protein (GFP). Given that repression of the p19ARF promoter is likely to be incomplete, we also tested whether removing one allele of p19ARF might enhance the ability of the IFP to induce leukemia by further reducing the expression of p19ARF. We found that expression of the IFP induced a myelomonocytic AML in 10 to 12 weeks when expressed in p19ARF-null cells. In addition, the IFP cooperated with the removal of only a single allele of p19ARF and the complete deletion of p19ARF only modestly shortened the latency period. The leukemic blasts in all the cases showed large nuclei and many of them contained granules and/or vacuoles in the cytoplasm. In addition, the GFP+ blast cells that express the IFP did not express any of the standard lineage markers and the majority of these cells were ckit+/Sca1−. All these findings are consistent with a myeloblastic acute leukemia. We conclude that repression of p19ARF by the IFP may stimulate formation of AML and that the IFP sets the stage for the rapid development of AML.