bluuebunny/crossref_metadata_2025 huggingface dataset:
Efficacy of Diverse High-Dose Regimens Followed by Autologous Peripheral Blood Stem Cell Transplantation in Consecutive Multiple Myeloma Patients: A Single Center Analysis over a 12 Year Period.
Authors: Haas Peter , Bauchmüller Kris , Kühnemund Alexander , Ihorst Gabriele , Engelhardt Monika
Published: April 2020

Over the last years, high-dose (HD) chemotherapy followed by autologous peripheral blood stem cell transplantation (auto-PBSCT) has emerged as one very effective approach to improve the outcome in multiple myeloma (MM) patients (pts). HD- regimens in MM have been Busulfan and Cyclophosphamide (Bu/Cy) and HD-Melphalan (MEL), with or without total body irradiation (TBI). In this analysis, we analyzed the efficacy of different HD-regimens in consecutive MM (pts) receiving an auto-PBSCT at our institution between 8/1992 and 1/2005. Within these 12.5 years, 120 MM pts (68 male, 52 female) received an auto-PBSCT. Their median age was 56 (27–74) years. According to Durie &Salmon (DS), stage I, II and III disease prior to PBSCT were observed in 5 (4%), 30 (25%) and 85 pts (71%), respectively. Bu/Cy was used until 1997, TBI/MEL between 1997 and 1999, and MEL alone thereafter. The respective HD-regimens consisted of Bu/Cy (Bu 16mg/kg; Cy 120mg/kg) in 15 pts (12.3%), TBI/MEL (10 Gy; MEL 140 mg/m2) in 16 pts (13.1%) and MEL (200mg/m2) in 89 pts (73.0%). The median OS of all MM pts after auto-PBSCT in our analysis was 59.5 months. The therapy related mortality (d0 – d+100 after PBSCT) was 0% (0/15) for Bu/Cy, 3.4% (3/89) for MEL alone and 6.3% (1/16) for TBI/MEL. Main Bu/Cy side effects were infections (n=4), pulmonary fibrosis (n=2), renal failure (n=1) and VOD (n=1). Infections, mostly fever of unknown origin, were observed in the MEL and TBI/MEL group in 68/89 and 15/16 pts, respectively, however in none, pulmonary fibrosis, renal failure or VOD occurred. Comparing Bu/Cy- vs. MEL-conditioning in terms of OS, Bu/Cy prolonged OS in some pts (77.2 vs. 55.6 months, p=0.12), however, this difference failed to be of statistical significance. Our analysis on the influence of TBI on MEL-conditioning revealed no advantage for the combination-therapy (55.6 (+TBI) vs. 59.5 months (−TBI)), which confirms previous studies, also show no benefit for the addition of TBI. In conclusion, our data verify that comparable remission and OS rates can be obtained with less toxic conditioning regimens in MM, such as MEL200 alone, thereby significantly reducing therapy-related side effects. Since auto-PBSCT is an effective treatment option also for elderly and frail MM pts, choosing the best conditioning regimen is important and should improve the treatment outcome in MM further. Current studies are assessing the use of tandem-transplantations and novel anti-MM-agents, such as Bortezomib, Revlimid, Thalidomide and others, before, with and after high-dose regimens. These treatment concepts make highly effective but less toxic conditioning regimens imperative for auto- and allo-transplantations.
High-Dose Sequential Chemotherapy Versus a Less Intensive Chemotherapeutic Regimen Followed by Peripheral Blood Progenitor Cell Autografting in Patients with Advanced Hodgkin’s Disease.
Authors: Nucci Marcio , Urago Katia , Miranda Eliana A. , Delamain Marcia , Souto Filho Joao T. , Matte Silvia F. , Duarte Carla M. , Oliveira Gislaine B. , Vigorito Afonso C. , Pagnano Katia B. , Maiolino Angelo , de Souza Carmino A.
Published: April 2020

High-dose chemotherapy (HDCT) followed by autologous stem cell transplantation (ASCT) is considered standard of care for patients with Hodgkin’s disease (HD) who relapse or progress after first-line chemotherapy. Recently, high-dose sequential chemotherapy (HDS) before ASCT has been evaluated, and phase II data suggest that it may improve the outcome without increasing significantly the toxicity. However, the lack of comparison between HDS and a more conservative strategy hampers any conclusion about the effectiveness of HDS. In this study, we compared in a non-randomized fashion, HDS (n=52) with a less-intensive chemotherapeutic regimen (non-HDS, n=60) followed by ASCT in patients with relapsed or refractory HD. Patients were treated at 2 Brazilian centers (HDS - UNICAMP; non-HDS - UFRJ). HDS consisted of cyclophosphamide (7 g/m2) followed by stem cell collection, methotrexate (8 g/m2) plus vincristine (1.4 mg/m2) and etoposide (2 g/m2), followed by BEAM and peripheral blood ASCT. Non-HDS patients received 2 cycles of DHAP, followed by cyclophosphamide (1,5 g/m2) followed by stem cell collection, followed by CBV and peripheral blood ASCT. There were more HDS patients with advanced disease (stage IV 40% vs. 13%, p=0.001), bulky disease (62% vs. 39%, p=0.02), and less patients in complete remission (CR) (6% vs. 18%, p=0.04). Toxic death before HSCT occurred in 5 patients in the HDS group and in none in the non-HDS group (p=0.02). Among 31 patients with progressive disease (PD) in the HDS group, 8 (25% remained in PD before ASCT, compared to 100% of 28 patients in the non-HDS group (p<0.001). The actuarial survival from HSCT was similar (58% in both groups, p=0.49), as was the disease free survival (64% in HDS vs. 80% in non-HDS, p=0.19). The outcome of patients from the HDS group who were in PD at the time of HSCT was significantly poorer than patients in CR or partial remission (actuarial survival zero vs. 77%, p<0.001). On the other hand, in the non-HDS group, the overall survival was not influenced by the disease status before ASCT (60% vs. 59%, p=0.77). Although HDS was associated with early deaths, it seemed to be beneficial to patients in PD. These results suggest that HDS could be preferably selected to patients with PD.
Long-Term Safety and Efficacy Data on Childhood Venous Thrombosis Treated with Enoxaparin: An Open Label Survey of Once-Daily Versus Twice-Daily Administration.
Authors: Nowak-Gottl Ulrike , During Christine , Bidlingmaier Christoph , Merkel Nick , Schobess Rosemarie
Published: April 2020

Low molecular weight heparin is as effective as unfractionated heparin for treatment of venous thromboembolism (VT) in adults, and it has been recently demonstrated that once-daily (q24h) or twice-daily (q12h) enoxaparin (E) administration had no impact on safety (bleeding rate) of efficacy (re-thrombosis rate) in the treatment of acute VT. However, in this setting it is still unclear for children, if enoxaparin should be administered q24h or q12h.

In this open label pilot safety study 75 children > the neonatal period, enrolled from two different catchment areas of Germany were treated with E with a target 2–4 h anti Xa activity between 0.4 – 0.8 IU/ml. After the acute thrombotic onset during which E was administered q12h for a median (range) period of 7 (1–14) days, based on the individual decision of the study centres stratification into the study arms once-daily versus twice daily was performed. Enoxaparin was administered in both study centres regardless of age at DVT-onset, thrombus burden, underlying basic diseases or presence of prothrombotic risk factors. No difference was observed between the stratifications performed in the study centres (p=0.5). Median (range) dose administered q24h (n=50) or q12h (n=25) in children with DVT was 1.2 mg/kg (0.5–3.0) respectively. Median (range) treatment duration was 5 months (1–13). Prospectively defined study endpoints were adverse effects, e.g. re-thrombosis, bleeding, and therapy-related death. Median follow-up time was 24 months.

No significant differences were found between the two patient groups treated with respect to anti Xa activities, efficacy and safety. Further statistical analysis performed to evaluate the overall differences between q24h and q12h E administration again revealed no significant differences for the study endpoints: Re-thrombosis occurred in two patients each (p=0.6), and a minor subdural bleeding was diagnosed in one patient treated q12h (p=0.4). During the follow-up no major bleeding or therapy-related death was observed.

In conclusion, data presented here give evidence that long-term enoxaparin administration within the reported target anti Xa activity for treatment of childhood VT has a good efficacy and safety profile when administered q24h. These data have to be confirmed in a multicentre randomised study.
Autologous Transplantation of Non-Cryopreserved Bone Marrow for the Treatment of High-Risk Adult Acute Lymphoblastic Leukemia in First Complete Remission.
Authors: Holowiecki Jerzy , Giebel Sebastian , Krawczyk-Kulis Malgorzata , Sadus-Wojciechowska Maria , Kachel Lucja , Wojnar Jerzy , Stella-Holowiecka Beata , Grosicki Sebastian
Published: April 2020

Autologous hematopoietic stem cell transplantation (HSCT) is a recognised option of post-consolidation therapy for adults with high-risk acute lymphoblastic leukemia (ALL) not having a donor. G-CSF-stimulated peripheral blood SCT results in faster recovery compared to cryopreserved bone marrow transplantation (BMT) and is currentlly used by the majority of centres. In the current study we analyze the feasibility of a new technique of autologous BMT, which does not require cryopreservation. 115 adult patients (median age 24.5 (16–53) years) with high-risk ALL in first complete remission (CR) were treated with autologous BMT between 1991–2004 in a single center using uniform standard operating procedures. Immune phenotype was as follows: proB 17%, preB 9%, common 44%, mature B 1%, preT 9%, mature T 19%. Initial WBC was >30 G/l in 30% of patients. 8% of patients were bcr/abl(+), 38% required >1 course of induction to achieve CR.

Bone marrow was collected in general anaesthesia and further stored for 72 hours in 4degC without any processing and reinfused 24 hours after completion of myeloablative therapy. Conditioning regimen (CAV) consisted of cytarabine 2x1000 mg/m2 d. −3, −2, −1, etoposide 800 mg/m2 d. −3, −2, cyclophosphamide 60 mg/kg d. −3, −2. Median NC dose was 2.0 (0.9–10.8)x10e8, CD34+cell dose − 1.6 (0.4–15)x10e6/kg. Median recovery of ANC>0.5 G/l equaled 16(11–45) days, PLT>50 G/l – 16(10–53) days (11% patients received cytokines to stimulate NC recovery). Median duration of hospital stay since the date of BMT was 19(13–51) days.

The OS rate at 10 years (median follow-up 6.5 years) equaled 57% (+/−5%), LFS rate − 47% (+/−5%). Three patients died within 100 days after ABMT of septic infections (non-relapse mortality rate − 2.6%). None of the analysed factors (age, WBC at diagnosis, immonophenotype, time to achieve CR) was found to influence the long-term outcome.

We conclude that autologous transplantation of non-cryopreserved bone marrow after CAV conditioning is feasible for adults with high-risk ALL. The method is characterized by fast recovery, short hospital stay and low non-relapse mortality, and may constitute good alternative to autologous PBSCT.
In Vivo Purging with Hyper-CVAD Plus Rituximab Followed by Autologous Stem Cells Transplant and Rituximab Maintenance in Newly Diagnosed Mantle Cell Lymphoma: A Pilot Study for GISL (Italian Cooperative Study Group for Lymphoma).
Authors: Benedetti Edoardo , Caracciolo Francesco , Galimberti Sara , Papineschi Federico , Pelosini Matteo , Cervetti Giulia , Castiglioni Maido Gioacchino , Mechelli Sandra , Scatena Paola , Brugiatelli Maura , Petrini Mario
Published: April 2020

Previously untreated mantle cell lymphoma (MCL) are consistently associated with poor prognosis when treated with CHOP-like regimens. Typically the CR rate is 20–30%, median FFS = 10–16 months and median OS = 3 years. In the attempt to improve outcome we used a high dose intensity regimen such as Hyper-CVAD (HCVAD) with autologous stem cell transplant (ASCT). Twenty patients entered the study but only 10 up to now are valuable. Patients were apheresed after 2nd course of HCVAD and if apheresis (LPH) were PCR positive (Bcl1+/JH+) a second set of LPH were performed after completion of 4th cycle. To perform an in vivo purging Rituximab 375 mg/m2 was added at day +1 and +9 after last dose of ARA-C; GCSF 10μg/kg was commenced on day +5 until LPH was ultimated. Rituximab maintenance (375 mg/m2 once weekly for 4 consecutive weeks) started 2 month post-ASCT and was repeated every 6 months. Ten patients have completed 4 HCVAD and 7 /10 underwent ASCT and were conditioned with BEAM. After 4 HCVAD 7/10 patients were in CR and 3/7 in PR. After ASCT 1 PR obtained a CR, 1 PD obtained a VGPR and 5 CR maintained CR. Only 4CR post ASCT have received Rituximab maintenance and maintain CR. Two patients (1 CR blastic variant and 1 PR) refused ASCT and after 4 HCVAD received Rituximab maintenance and both are in CCR. Overall with a median follow up of 28.6 months (range 12–51) median survival is not reached. At 4 years 77.8% of patients are alive and PFS is 87.9%. Patients were monitored for bone marrow-MRD by PCR. Eight out of 10 were PCR+ at diagnosis and 7/8 were PCR negative after 4 HCVAD. After ASCT one PCR+ converted to PCR- and 1 PCR+ patient after 4 HCVAD converted to PCR- with Rituximab maintenance (refused ASCT).

Conclusions: high dose intensity regimen HCVAD + Rituximab as in vivo purging and for maintenance allowed to collect tumor free grafts in 70% of PCR+patients at diagnosis and to reach an ORR of 100%, 7/10 CR and PR 3/10 (included 1 blastic variant). PCR negativity was obtained in 7/8 patients. One patient from PR converted to CR after ASCT and one after Rituximab maintenance (without ASCT); thus we might speculate that both high doses (BEAM) and Rituximab do play a role to increase the probability to obtain a CR and PCR-. Survival and PFS of 77.8% and 87.9% respectively at 4 years are encouraging. Further follow up and a higher enrolment of patients are needed to better define the role of HCVAD + Rituximab and ASCT to increase CR,PCR- PFS and OS in MCL.
Comparison of the Peripheral Blood Stem Cell Mobilization and Harvest after Each Consolidation Chemotherapy in Patients with Acute Myeloid Leukemia in First Complete Remission Who Underwent Autologous Stem Cell Transplantation.
Authors: Shin Ho-Jin , Chung Joo Seop , Choi Young Jin , Lee Je-Jung , Sohn Sang Kyun , Cho Goon Jae , Kim Hyeoung-Joon , Yang Deok-Hwan , Back Jin Ho
Published: April 2020

Background: Peripheral blood stem cells (PBSC) are increasingly being used to restore hematopoiesis following high-dose chemotherapy in patients with acute myeloid leukemia (AML) without an HLA-matched related donor. However, which of consolidation-mobilization chemotherapy cycles are the most compatible in terms of the yield of progenitor cells and outcomes after autologous peripheral blood stem cell transplantation (APBSCT) has not been known.

Patients and methods: 49 mobilization procedures performed on 28 patients with AML who underwent APBSCT enrolled at three centers. The treatment protocols administered to patients with AML consisted of an induction chemotherapy in combination with cytarabine or BHAC plus idarubicin, and consolidation chemotherapy with high dose cytarabine (3 g/m2 iv bid, day 1, 3 and 5). In all patients, the collection of PBSC was performed during recovery after giving consolidation chemotherapy and granulocyte colony-stimulating factor (G-CSF).

Results: 28 AML patients in first complete remission (CR) underwent total 49 aphereses to obtain a minimum dose of 2×106/kg CD34+ cells/kg after each consolidation chemotherapy. First, second and third consolidation cycles were 17, 24 and 8, respectively. According to the total collected CD34+ cells ×106/kg, two main groups were considered that is, poor mobilizers (PM), with a collection of < 2×106/kg, and good mobilizers (GM), with a collection of ≥ 2×106/kg. Of 49 consolidation cycles, 15/17 (88.23%), 16/24 (66.66%) and 3/8 (37.5%) were GM after 1st, 2nd and 3rd consolidation cycles, respectively (P=0.012). The median follow up was 22 months (range 3–59). PBSC mobilization after first plus second versus third consolidation chemotherapy that was used for APBSCT were compared. The 2-year disease-free survival (DFS) rate was 36.1% and 64.3% (P=0.161), and the 2-year overall survival (OS) rate was 34.9% and 80% (P=0.190). Patients who were infused with ≥ 2 ×106/kg CD34+ cells at APBSCT had a shorter DFS rate at 2 years, 25.0%, than those with < 2 ×106/kg CD34+ cells, 53.8%. However, there were no significant differences between the two groups (P=0.169). Relapse rate after stem cells infusion which mobilized from first, first plus second, second and third consolidation cycles were 60% (3/5), 61.1% (11/18), 50% (4/8) and 2/6 (33.3%), respectively.

Conclusion: If reinfused CD34+ cells are adequate, the stem cells harvested after third consolidation chemotherapy may be the best source for APBSCT in view of relapse rate and DFS.
Use of Pharmacogenetics and Clinical Factors To Predict the Maintenance Dose of Warfarin.
Authors: Gage B. F. , Eby C. S. , Johnson J. A. , Rieder M. J. , Ridker P. M. , Rettie A. E. , Aquilante C. , Milligan P. E. , Marsh S. , Voora D. , Langaee T. , Veenstra D. L. , Birman-Deych E. , Glynn R. , Nickerson D. A. , McLeod H. L.
Published: April 2020

Background Initiation of warfarin therapy using trial-and-error dosing can cause bleeding. Clinical factors explain only 20%–30% of the variability in the therapeutic dose of warfarin. Single nucleotide polymorphisms (SNPs) in the cytochrome P450 2C9 (CYP2C9) gene correlate with the clearance of S-warfarin and SNPs in the vitamin K epoxide reductase (VKORC1) gene predict warfarin sensitivity. We test the hypothesis that the combination of clinical and pharmacogenetic information can predict the therapeutic warfarin dose.

Methods We collected DNA, demographic variables, laboratory values, and medication histories from patients taking warfarin. Subjects either attended an outpatient anticoagulation clinic or participated in the PREVENT (prevention of venous thromboembolism) study. After PCR amplification, we used Pyrosequencing® to genotype DNA regions for 2 coding CYP2C9 SNPs, *2 (C430T) and *3 (A1075C), and for 4 noncoding VKORC1 SNPs: C861A, A5808C, G6853C, and G9041A. Using multiple regression, we quantified the association between therapeutic warfarin dose and clinical and genetic factors in a derivation cohort of 900 participants and a validation cohort of 100 participants.

Results The VKORC1 G6853C SNP was the first variable to enter the stepwise regression equation and was associated with a 27% decrease in the warfarin dose per allele in Caucasian patients. The VKORC1 A5808C SNP was associated with a 33% decrease per allele in warfarin dose in African-American patients. Other significant (p < 0.05) predictors of the therapeutic warfarin dose, in order of entry into the regression equation and their effect on warfarin dose were: body surface area (+12% per SD increase), CYP2C9*3 (−33% per allele), CYP2C9*2 (−20% per allele), age (−7% per decade), target INR (+8% per 0.5 unit increase), amiodarone use (−24%), African-American race (+12%), smoker (+9%), and simvastatin or fluvastatin use (−5%). A dosing equation that included these pharmacogenetic and clinical factors explained 52% of the dose variability in derivation cohort and 55% of the variability in the validation cohort.

Conclusions The therapeutic warfarin dose can be estimated from clinical and pharmacogenetic factors that can be obtained when warfarin is started. Use of this dosing equation has potential to aid in the prediction of an optimal warfarin dose, which may decrease the risk of bleeding during the initiation of warfarin therapy.
Effective CD34+ Stem Cell Mobilization with Low-Dose Pegfilgrastim.
Authors: Gladstone Douglas E. , Stevens Michelle , Locher Emily , Zamkoff Kenneth W. , Hagag Nabil G.
Published: April 2020

Introduction: Numerous strategies utilizing chemotherapy followed by filgrastim allow for the successful mobilization of peripheral hematopoietic progenitor cells. To eliminate the need for self-administration of multiple growth factor injections and to minimize cost, we piloted a mobilization strategy consisting of low-dose Pegfilgrastim and chemotherapy.

Methods: Seven consecutive patients scheduled to undergo hematopoietic progenitor cell transplant for hematologic malignancy received a single 6mg injection of Pegfilgrastim 24–48 hours after chemotherapy. The targeted CD34+ collection was 5x106cells/ kg. The number of apheresis, CD34+ cell yield, time to engraftment and toxicities were recorded. These patients were compared to the 15 previous patients who received Filgrastim and chemotherapy for mobilization.

Results: In the pilot population, the median age was 60 (range: 39–67) years. Chemotherapy priming before Pegfilgrastin for three patients with multiple myeloma and two with stage IV mantle cell lymphoma was 4.5grams/m2 of cyclophosphamide; one patient with relapsed, stage IV Hodgkin’s disease received ICE and one multiple myeloma patient with a serum creatinine of 3.1mg/dL, received 2.5grams/m2 of cyclophosphamide. Five patients required a single apheresis, two patients required two apheresis collections. The median CD34+ yield was 6.7 x106/kg (range: 3.2–13.7). The median time to neutrophil engraftment was 10 days (range: 9–12). The median time from Pegfilgrastim to apheresis was 9 days (range: 7–10). The Pegfilgrastim cost per patient was $3,250.00. Toxicity was limited to grade II bone pain.

The comparison group consisted of seven multiple myeloma, four leukemia and four lymphoma patients. Their median age was 51.5 years (range: 24–67). The median number of apheresis procedures was 2 (range: 1–3). The median CD34+ yield was 5.6 x106/kg (range: 2.9–17.6). The median time to neutrophil engraftment was 11 days (range: 9–11). The median time from cytokine to apheresis was 8.5 days (range: 3–16). The median cost of Filgrastim per patient was $3,650.00 (range: $2,290–$8,760). The median number of Filgrastim injections was 10 (range: 10–14). Toxicity was limited to grade II bone pain.

Discussion: In a two cohorts of patients with hematologic malignancies Pegfilgrastim as compared to Filgrastim resulted in a decreased cytokine cost, a fewer number of apheresis collection procedures, a more predictable time to first apheresis and no self-administration of medication. There was no significant difference in the time to engraftment.

Conclusion: Low-dose Pegfilgrastim in conjunction with chemotherapy results in as effective CD34+ progenitor cell mobilization with less drug cost and apheresis time as compared to Filgrastim with chemotherapy.
Tandem Autotransplants’ Peripheral Blood Stem Cells Following Sequent High-Dose CHEOP Chemotherapy for Aggressive Lymphoma.
Authors: Luo Yi , Huang He , Cai Zhen , Lin Maofang
Published: April 2020

The majority of poor-risk lymphoma patients are not cured with conventional chemotherapy. There is evidence for superiority of single high-dose chemotherapy in such patients, but many still die from recurrent disease. Strategies to improve survival in these poor-risk patients include dose-intensification with high-dose chemotherapy and PBSC support autologous transplantation. These more aggressive strategies are feasible and tolerable. Whether tandem transplantation will prove more effective than current single high-dose therapy in appropriately selected patients remains to be determined. The purpose of this study was to evaluate the effectivity and safety of mobilization regimen, and the effectivity and tolerance of sequential chemotherapy combined with tandem autotransplant in aggressive lymphoma. In this study, the clinical data of 5 patients with recurrent, aggressive lymphoma treated with of sequential chemotherapy combined with tandem autotransplant were analysed respectively. The group included 1 HD and 4 NHL. Mobilization regimen was CHOEP combined with G-CSF (5ug•kg−1•d−1). The conditioning regimen for the tandem transplantation was high-dose CHEOP. The interval of the two autotransplantation was 9(5~31)weeks. In tandem autotransplant, the cell number of MNC which was transfused was 3.05 (ranged from 1.91~4.14) ×108 •kg−1 and 3.55 (ranged from 2.23~6.0) ×108•kg−1; CD34+ was 4.11 (ranged from 2.59~4.94) ×106•kg−1 and 5.70 (ranged from 2.77~10.6) ×106•kg−1; CFU-GM was 2.96 (ranged from 2.01~4.54) ×105•kg−1 and 2.44 (ranged from 1.78~2.9) ×105•kg−1 respectively (P>0.05). All patients reached prompt and sustained hemotopoietic reconstitution. The interval of ANC ≥0.5×109/L was 10 days (ranged from 8~12) and 10.5 days (ranged from 9~12); Pt ≥2.0×109/L was 11 days (ranged from 10~14) and 12.5 days (ranged from 10~15) respectively (P>0.05). Four patients were alive while 3 was in disease-free with median 46 (range 9~88) months. The overall survival was 80%, and the disease-free survival was 60%. In the conclusion, the method of sequential high-dose CHEOP chemotherapy combined with tandem autotransplant of peripheral blood stem cells for aggressive lymphoma is safe and effective.
Natural History of Relapse after Autologous Stem Cell Transplantation for Multiple Myeloma.
Authors: McClune Brian , Buadi Francis , Yunus Furhan , Minhas Sohail , Walsh William , Przepiorka Donna
Published: April 2020

Although autologous stem cell transplantation (APBSCT) is known to prolong survival of patients with myeloma, most patients relapse after transplantation. How treatment of patients who relapse after transplantation affects survival is not clear. At our institution, of 83 patients transplanted for myeloma from 1996–2005, we identified 25 patients who relapsed and had adequate records for analysis. The relapse cohort consisted of 15 single APBSCT and 10 tandem APBSCT recipients. Twenty patients were Stage III at time of original diagnosis, 3 patients were Stage II, and 3 were Stage I with 15 patients being IgG-kappa, 3 patients IgA-kappa, 2 patients IgG-lambda and 1 patients IgA-lambda. Two patients had non-secretory disease and 2 had light-chain only myeloma. Best response achieved after transplant included 5 complete responses (CR), 14 partial responses (PR), one stable disease (SD), 3 minimal responses (MR), and 2 with progressive disease (PD). Maintenance therapy consisting of interferon-alfa, pulse-dosed or every-other-day steroids, and thalidomide with or without steroids was documented in 12 patients. Of the relapsed cohort, 19 went on to receive further treatment consisting of radiation therapy (2), dexamethasone-based chemotherapy [bortezomib, another induction regimen, thalidomide] (15) or another transplant (2). The median duration of response for the relapse cohort was 372 days (76–1661 days). Median survival from relapse is 304 days (range 19–1155 days). There was a trend for a correlation between duration of initial response and survival after relapse (p=0.08), but no correlation with survival after relapse and best response to APBSCT (p=0.63), use of maintenance posttransplant (0.27) or treatment of relapse (0.17). The small number of patients may have limited the power of our study to detect significant associations in our cohort. We conclude that patients who relapse after APBSCT can have a prolonged survival, and those with the longest initial duration of response have the best outcome, but the optimal approach to salvage for these patients is not clear.
POEMS Syndrome: A Patient with an IgA lambda Plasma Cell Disease. Expression Levels of VEGF and bFGF Correlates with Clinical Course.
Authors: Schrader Carsten , Tiemann Markus , Zirrgiebel Ute , Guenther Andreas , Janssen Dirk , Kneba Michael , Gramatzki Martin
Published: April 2020

Purpose: POEMS syndrome is a rare disease characterized by polyneuropathy, organomegaly, endocrinopathy, M-protein and skin changes. We present a severely ill woman with a four year history of polyneuropathy showing all signs of a POEMS syndrome. Response to chemotherapy including high-dose melphalan treatment and autologous bone marrow transplantation was monitored and vascular endothelial growth factor (VEGF) as well as basic fibroblast growth factor (bFGF) levels were measured.

Methods: Blood investigation was done for serum electrophoresis analysis and analysis of VEGF, bFGF and IL-6 by ELISA. Bone marrow biopsy specimen was investigated immunohistochemically for kappa, lambda, alpha, gamma, CD20, CD56, Cyclin D1, VEGF.

Results: Immunohistochemical investigation of the bone marrow biopsy showed a infiltration of lambda and alpha positive plasma cells (10%). Only few plasma cells expressed kappa. The tumor cell were negative for CD20, CD56 and Cyclin D1, but positive for VEGF in line with the high VEGF levels in the blood. Blood investigation revealed a discrete monoclonal gammopathy of IgA lambda type. Initially, high levels of VEGF (1468.7 pg/ml) and bFGF (112.9 pg/ml) were detected. However, treatment with high-dose melphalan and tandem autologous bone marrow transplantation proved extremely helpful in symptom control. Already after the first transplant the patient started to walk again and lost pulmonary hypertension. In parallel VEGF and bFGF levels decreased and the performance status of the patient improved dramatically.

Conclusion: VEGF and bFGF measurement is a useful tool for monitoring disease activity in POEMS syndrome. Bone marrow transplantation is an important therapy also in severely ill patients.
Idiotype TCR Vβ2 DNA Plasmid Constructe, Transfer and Express in K562 Cells.
Authors: Zhou Yubing , Huang Meijuan , Yang Lijian , Chen Shaohua , Wu Xiuli , Li Yangqiu
Published: April 2020

Recently, combination of different therapeutic strategies have significantly increased survival in patients with hematologic malignancies. Specific immunotherapy is an important anticancer therapy to eradicate minimal residual disease in leukemia patients after chemotherapy or stem cells transplantation. DNA vaccines have been showed to induce strong and persistent cell-mediated and humoral immune responses and used in Hodgkin lymphoma patients, using the idotype Ig antigen. In order to develop the anti-lymphoblastic leukemia idiotypic TCR DNA vaccine, which was expected to induce the specific immune response anti T-cell lymphoblastic lymphoma /leukemia in vivo. The rearranged idiotypic CDR3 fragment coding TCR Vβ2, which was identified from a TCR Vβ2 clone-Molt4 cell line, was amplified using RT-PCR, and the PCR products were then cloned into pIRES vector. The recombinant plasmids contaning target gene (405 bp, 135 peptides) were analyzed by digestion with restriction enzyme (EcoRI and XbaI), PCR and sequencing. The correct fragment was transfected into K562 cells. The condition of idiotypic protein expression was tested by indirect immunophenotyping fluorescein dyeing with anti-TCR Vβ2 monoclonal antibody, SDS-PAGE and Western-Blot. The results showed that the recombinant DNA plasmids, pIRES-Molt4 Vβ2, containing idiotypic TCR Vβ2 frgments of the Molt4 cell line were developed successfully. A 15 KD protain which can bind with TCR Vβ2 antibody specially were identified from pIRES-Molt4 Vβ2 transduced K562 cells, indicating that can express special TCR Vβ2 protain in vitro. It should be further demonstrated whether the idiotype protein can elicit both humoral and cellular immune response for anit Vβ2+ leukemic cells in vivo.
Interaction of Measles Virus Vectors with Auger Electron Emitting Radioisotopes.
Authors: Dingli David , Peng Kah-Whye , Harvey Mary E. , Vongpunsawad Sompong , Bergert Elizabeth R. , Kyle Robert A. , Cattaneo Roberto , Morris John C. , Russell Stephen J.
Published: April 2020

Background: Viral vectors based on the Edmonston strain of measles virus (MV-Edm) selectively destroy all tumor cell lines tested in vitro. The oncolytic activity of the virus is enhanced by expression of the thyroidal sodium iodide symporter (MV-NIS) that allows selective 131I uptake by infected tumor cells and eliminates myeloma tumor xenografts that are resistant to the parent virus. MV-NIS is being considered for therapy of patients with relapsed or refractory multiple myeloma. Advanced myeloma is associated with significant immunosuppression with the potential risk of uncontrolled virus proliferation. The number of agents with activity against MV is limited. Low energy (Auger) electrons have a short path length and selectively damage cells in which the isotope decays. Thus, we hypothesized that the Auger electron emitting isotope 125I, selectively taken up by cells expressing NIS, can be used to control viral proliferation.

Methods: A replication competent MV that expressed both a soluble form of carcinoembryonic antigen (CEA) and NIS (MV-NICE) was rescued and characterized. Cells were infected with MV-NICE or control vectors and exposed to 125I with appropriate controls. CEA expression and viral titers were determined at different time points. The role of free radical generation on virus replication was explored. In vivo control of MV-NICE replication with 125I was attempted.

Results: MV-NICE replication in vitro is inhibited by the selective uptake of 125I by cells expressing NIS. Extracellular decay of the isotope has no effect on virus proliferation. Auger electron damage is in part mediated by free radicals and abrogated by glutathione. In myeloma xenografts, control of MV-NICE with 125I was not possible under the conditions of the experiment.

Conclusion: MV-NICE does not replicate faster in the presence of radiation under our experimental conditions. Auger electron emitting isotopes effectively stop propagation of MV vectors expressing NIS in vitro. Additional work is necessary to translate these observations in vivo.
Automated Retroviral Insertion Site Sequence Analysis and Mapping Tool Followed by Database Analysis.
Authors: Laufs Stephanie , Giordano Frank A. , Hotz-Wagenblatt Agnes , Appelt Uwe , Lauterborn Daniel , Fellenberg Kurt , Zeller W. Jens , Fruehauf Stefan
Published: April 2020

Increasing use of retroviral vector-mediated gene transfer and recent reports on insertional mutagenesis in mice and humans created intense interest to characterize vector integrations on the genomic level. Techniques to determine insertion sites, mainly based on time consuming manual data processing and compilation, are thus commonly applied in gene therapy laboratories. Since a high variability in processing methods hampers further data comparison, there is an urgent need to systematically process the data arising from such analysis.

The obtained sequences from the integration site analysis are judged to be authentic only if the matching part of the genomic query sequence is surrounded by the 5′LTR-sequence on the one side and the adapter-sequence on the other side. Therefore we developed an Integrationseq tool. In this task, different methods for converting the ABI sequence trace files to high quality sequences and for recognizing and deleting the LTR and adaptor parts of the isolated clones were implemented. If neither a primer nor a LTR could be found, the sequence is discarded. If the LTR is found on the complementary strand, the integration sequence is reversed. The remaining sequence between primer and LTR positions are taken as the n integration sequence and written to a sequence output file.

We validated the Integrationseq tool using 259 trace files originating from integration site analysis (LM-PCR). Sequences can be trimmed by IntegrationSeq, leading to an increased yield of valid integration sequence detection, which has shown to be more sensitive (100%) than conventional analysis (94.3%) and 15 times faster than conventional analysis, while the specifities are equal (both 100%). Valid integration sequences get further processed with IntegrationMap for automatic genomic mapping. IntegrationMap runs 50 times faster than conventional methods and retrieves detailed information about whether integrations are located in or close to genes, the name of the gene, the exact localization in the transcriptional units and further parameters like the distance from the transcription start site to the integration. Further information, e.g. data about CpG-Islands, LINEs or SINEs, and their distances to the integration is also displayed. Output files generated by the task were found to be 99.8% identical with results retrieved by conventional mapping with the Ensembl alignment tool.

Using both tools, IntegrationSeq and IntegrationMap, a validated, fast and standardized high-throughput analysis of insertion sites can be achieved for the first time.
RITA Activates p53 Function in Chronic Lymphocytic Leukaemia Cells and Induces Apoptosis.
Authors: Nahi Hareth , Merup Mats , Möllgård Lars , Selivanova Galina , Paul Christer
Published: April 2020

The tumor suppressor gene p53 is the most commonly mutated gene in solid tumors. Although less common in hematologic malignancies, about 10–20% of CLL cases carry a p53 mutation. Recently, the compound RITA (Reactivation of p53 and Induction of tumor cells Apoptosis) has been shown to able to bound to p53 and induce its accumulation in tumor cells, its seems to have a cytotoxic effects in tumors with wild type p53.

Peripheral blood or bone marrow was collected from 17 patients with B-CLL, 6 samples wit deleted p53 and 11 without. The cryopreserved samples were incubated in 2 and 3 days with RITA and fludarabine alone or RITA and fludarabine in combination. The cells (3.0x105 cells/ml) were incubated in a medium consisting of RPMI 1640, glutamax, 10% PBS (Phosphate Buffered Saline). Cell viability was assessed after incubation by a bioluminescence method measuring cellular ATP. RITA induced dose and time dependent cell toxic effects in all B-CLL cells but was most effective in those with wild type p53. The combination of 10μM RITA and 1μM of fludarabine resulted in a very high significant synergistic effect. RITA induced dose dependent cell toxic effects in both B-CLL cells with p53 mutation and those without. In the non mutated cells, the main viability compared to unexposed controls after two days of incubation was 54 % (range 20–88 %) at 10μM, 42 % (13–83 %) at 20μM and 31 % (6–870 %) at 50μM. In the patient with a confirmed p53 mutation, the cell viability values were 70 %, 56 % and 30 %, respectively. The combination of 10μM RITA and 1μM of fludarabine decreased the cell viability with an average of 56 percentage points compared to fludarabine alone. We conclude that RITA induces cytotoxic effect in B-CLL and that CLL cells without p53 deletion are more sensitive in vitro. (Preliminary results suggest that RITA cause accumulation of p53 and cause massive apoptosis) Determination of p53 status and apoptosis in the tested patients before and after exposure to RITA is currently performed and the results will be presented at the meeting.
A New Human CD20 Antibody for Improved Killing of CD20-Transgenic T Cells for Allogeneic Stem Cell Transplantation.
Authors: van Meerten Tom , Mackus Wendy J.M. , Parren Paul W.H.I. , van de Winkel Jan G.J. , Hagenbeek Anton , Ebeling Saskia B.
Published: April 2020

Allogeneic stem cell transplantation together with donor lymphocyte infusion represents the best curative option for many haematological malignancies. Donor T lymphocytes play a central role in the elimination of leukemic cells (Graft versus Leukemia, GvL) and acceptance of the graft. Unfortunately, these donor T cells also respond to the patients’ own tissue, resulting in Graft versus Host Disease (GvHD).

In a promising strategy to control GvHD, while at the same time maximally utilizing the GvL effect, genetically-modified donor T cells, expressing a suicide gene, are selectively eliminated in vivo in case of severe GvHD. Genetic transfer of the human CD20 molecule to donor T cells prior to transplantation permits selective elimination via CD20-reactive reagents, such as the widely used chimeric Ab rituximab (RTX). RTX activates complement (CDC), and recruits effector cells (ADCC) for elimination of CD20-positive cells.

Using a novel experimental system we previously showed that the efficiency of RTX-mediated killing of CD20-positive T cells clearly depends on the level of CD20 expression. As a consequence, RTX is not able to kill all genetically modified CD20+ T cells.

Here we investigated whether a novel fully human CD20 Ab, HuMab 7D8, would be better in activating complement and/or effector cells. Our results show HuMab 7D8 to be more powerful in activating complement in CDC-assays. To obtain a similar percentage cell kill compared with RTX, a 100-fold lower dose of HuMab 7D8 was needed and a 4 times lower human serum concentration. To study the effect of the number of CD20 molecules per cell we used CEM-CD20 clones (N=26), all with different CD20-expression levels, in an assay in which all other parameters were kept constant. The results show that HuMab 7D8 is significantly stronger in inducing CD20-mediated cell kill compared to RTX. Especially cells expressing very low levels of CD20, which are completely resistant to RTX-induced CDC, are lysed with high efficiency by HuMab 7D8 and complement. In contrast to RTX, HuMab 7D8 was able to eliminate 100% of transduced T cells in the presence of human serum, and effector cells. At present, studies with CD20-luciferase positive human T cells are conducted in mice to study the in vivo effect of HuMab 7D8 and RTX. These results demonstrate that the CD20 molecule represents an attractive suicide molecule and CD20-transgenic T cells can with high efficiency be eliminated by HuMab 7D8.
Genetically Modified Her2-Specific T Cells Recognize Low and High Her2 Expressing Breast Cancer Cells.
Authors: Ahmed Nabil M. , Ratnayake Maheshika , Pule Martin , Rooney Cliona M. , Heslop Helen E. , Gottschalk Stehpen M.
Published: April 2020

Background: Only patients with breast cancers expressing high levels of Her2 benefit from anti-Her2 monoclonal antibodies such as trastuzumab. In the present study we investigated in vitro, if chimeric T-cell receptors (TCR), which combine the antigen-specificity of monoclonal antibodies with the effector function of T cells can overcome this limitation.

Material and Methods: T cells from healthy donors were transduced with retroviral vectors containing the Her2-specific chimeric TCR gene with a CD28-zeta signaling domain (Her2-CD28-zeta). The specificity of the genetically modified T cells was determined by their ability 1) to kill breast cancer cell lines in cytoxicity assays, and 2) to proliferate and secrete cytokines (IFN-γ and IL-2) after stimulation with breast cancer cell lines. The following panel of cell lines was used: autologous PHA blasts, MDA-MB-468 (both Her2-negative), MCF-7 (Her2-low), Her218 and SKBR-3 (both Her2-high).

Results: Her2-CD28-zeta expressing T cells killed low and high Her2-positive breast cancer cell lines in cytotoxicity assays, where as Her2-negative T cells were not killed. Stimulation of T cells with breast cancer cell lines expressing both high and low levels of Her2 resulted in T-cell proliferation and secretion of IFN-γ and IL-2 in a HER2 dependent manner.

Discussion: We demonstrate that breast cancer cells with low levels of expression of Her2 can effectively activate T cells expressing Her2-specific chimeric T cell receptor, induce T-cell proliferation, and the production of IL-2, an important T-cell growth factor. These results indicate that T cells expressing chimeric TCRs could possibly extend the application of Her2 targeted therapies to malignancies expressing low levels of Her2.
Efficacy of Zymogen Factor VII Is Comparable to Activated Factor VII for Nonviral Gene Therapy Treatment of Hemophilia A.
Authors: Chen Steven C. , Greenberg Daniel , Ye Peiqing , Davie Earl W. , Miao Carol H.
Published: April 2020

A major problem for clinical treatment of hemophilia A using factor replacement therapy is the high frequency formation of inhibitory antibodies against factor VIII. This problem is also predicted to occur following strategies currently aimed at targeted genetic correction of this disease. Recombinant activated factor VII (rFVIIa, NovoSeven®, Novo Nordisk, Bagsvaerd, Denmark) has been successfully used as an effective alternative treatment for hemophilia patients who have developed inhibitors. In order to decrease the cost and fluctuation of FVIIa levels associated with frequent infusions of rFVIIa, nonviral gene transfer of factor VII (FVII) was attempted in a hemophilia A mouse model. In addition, to investigate the potential thrombotic risks associated with prolonged, high level of FVIIa expression following gene transfer, we compared the effects of gene transfer vectors encoding zymogen FVII with an engineered secreted FVIIa in hemophilia A mice. We inserted murine FVII (mFVII) cDNA into a liver-specific vector developed recently in our lab to generate pBS-HCRHPI-mFVII-A. The mFVII cDNA sequence was then modified by site-directed mutagenesis to insert a protease cleavage site in between Arg152-Ile153. The resulting construct of murine FVIIa (mFVIIa) encodes a modified protein, which can be cleaved by intracellular proteases of the furin family to secrete the activated form of mFVII. Fifty mg of either mFVII or mFVIIa the plasmid was delivered into hemophilia A mouse liver by a rapid, high volume (hydrodynamics-based) infusion method (n=8 mice/group). Phenotypic correction was evaluated using tail-clip assays by measuring the hemoglobin levels in the collected blood. This assay indicated that vectors carrying either mFVII or mFVIIa can partially correct clotting following gene transfer. Significant correction in PT and APTT was observed in mFVII or mFVIIa-treated mice. Plasma-based and platelet-based thrombin generation (TG) assays indicated that either mFVII or mFVIIa significantly shorten peak time and peak thrombin levels of TG. In addition, these effects lasted long-term in plasmid treated mice for at least three months (experimental duration). We also constructed vectors carrying human FVII (hFVII) or FVIIa (hFVIIa) to test their efficacy for correcting hemophilia A. Constructs produced supra-physiological levels (800–1200ng/ml) of hFVII or hFVIIa assayed by ELISA at days 1 and 4 post-injection. However, the levels of hFVII and hFVIIa dropped precipitously at day 14 post-injection shown by both ELISA and Western blot, most likely due to the formation of species-specific antibodies against hFVII. These results clearly demonstrate that nonviral gene therapy of either zymogene FVII or activated FVII can rescue bleeding in hemophilia A mice.