bluuebunny/crossref_metadata_2025 huggingface dataset:
Development of a Potency Assay for Umbilical Cord Blood.
Authors: Reems Jo Anna , Hall Karen M. , Gebru Luluday H. , Rich Ivan N.
Published: October 2019

Total nucleated cell counts, cell viability, CD34 cell numbers and the colony-forming cell (CFC) assay constitute the main parameters used to characterize the quantity and quality of progenitors present in a unit of umbilical cord blood (UCB) prior to transplantation. Of these parameters, only the CFC assay provides information about the functional potential of progenitors. However, technical difficulties associated with the CFC assay have made it difficult to standardize this method among various UCB banks. Consequently, we are focusing on the development of a novel assay that relies on determining the growth potential of progenitors in response to cytokines by measuring intracellular ATP levels (HALO SPC-QC, Hemogenix). This assay is based on the principle that cytokine stimulated proliferation responses by hematopoietic progenitors are proportional to the production of intracellular ATP. To perform the HALO assay, cells obtained from leukocyte enriched UCB units are added to cytokine impregnated methylcellulose media and deposited in replicates of 6 into 96-well plates. After 7 days of culture, cells are lyzed to release ATP, which drives a luciferin/luciferase reaction producing bioluminescence. ATP levels for each well are measured in a plate luminometer and the assay is standardized using an ATP control. Since proliferation, not differentiation is measured, the assay is completed in 7 days rather than the 10–14 days required for CFC assays. For mononuclear cells or erythrocyte-depleted cord blood, ATP levels for cell concentrations as low as 750 cells/well can be detected. An intra-laboratory study comparing total mean ATP (μM) production/well (6 replicates) to the CFC counts from mononuclear cells from UCB, showed a correlation coefficient of 0.99 (n=6). An inter-laboratory study using aliquots from the same UCB to compare the HALO and CFC assays at 2 geographically separate sites revealed correlation coefficients of 0.90 and 0.34 (n=10), respectively. Together these data indicate that the HALO assay is capable of measuring the clonogenic potential of UCB progenitors. These data also demonstrate that highly reproducible results can be achieved by different testing sites when measuring ATP production from proliferating progenitors. Thus, we suggest that the HALO method may be more conducive to standardization among UCB banks. We also propose that the HALO assay may be better than the CFC assay for accurately and precisely measuring the potency of an umbilical cord blood unit prior to transplantation.
Durable Engraftment with Minimal Toxicities after Allogeneic Hematopoietic Stem Cell Transplantation with a Reduced-Intensity Regimen Incorporating Fludarabine and 8 mg/kg Busulfan.
Authors: Uchida Naoyuki , Fukuda Takahiro , Wake Atsushi , Kim Sung-Won , Masuoka Kazuhiro , Mori Shin-ichiro , Miyakoshi Shigesaburo , Tanosaki Ryuji , Takaue Yoichi , Taniguchi Shuichi
Published: October 2019

Background: The optimal regimen for allogeneic stem cell transplantation with a reduced-intensity conditioning regimen (RIST) needs to be established. Therefore, we retrospectively reviewed data of 84 patients who underwent RIST using 180 mg/sqm fludarabine and 8 mg/kg busulfan (Flu/Bu8). All patients received mobilized blood stem cells from a related donor between Nov. 2000 and Jun. 2005.

Patients and Methods: The median age of the patients was 53 years (range, 25–65). The diagnosis included 42 AML/MDS, 34 lymphoma and 8 others. Sixty-nine patients (82%) had high-risk diseases, including 56 (81%) in non-remission status before RIST. All were serologically 6/6 matched pairs, except for one who had a 5/6 match. For GVHD prophylaxis, 61 used cyclosporine A (CSP) alone, 21 used CSP + methotrexate (MTX), and 2 used some other compounds.

Results: The engraftment kinetics were remarkable since all of the patients successfully engrafted at a median of 11 days (5–24 days), with no late graft rejection. Toxicities of grade 2 or 3 according to CTCAE v.3 were observed at minimal frequencies, i.e. 38 mucosal, 35 hepatic, 13 GI tract, 12 renal, 2 cardiac, 1 pulmonary, 19 febrile neutropenia, and 9 documented infections, and there were no grade 4 toxicities. The administration of MTX increased the incidence and severity of stomatitis (P=0.05). The cumulative incidences of grade II–IV and III–IV acute GVHD were, respectively, 50% and 29%, and 76% experienced chronic GVHD. Seventy-three patients (87%), including 38 of the 56 patients who were not in CR at the time of transplant, achieved partial or complete remission. The median follow-up of surviving patients was 1349 days (34–1981 days), and the day-100 mortality was 11% (disease progression 6% and non-relapse mortality [NRM] 5%). The 5-year overall and progression-free survival rates were 36% and 30%, respectively. Among 46 total deaths, 27 were from disease progression and 19 were from NRM primarily related to GVHD. The patients in remission at transplant showed better overall survival than those who were not in remission (85 % vs 34 % at 5 years post-transplant, respectively, P<0.05). The 5-year overall survival was 85 % for those in remission at transplant and 34 % for the remaining patients (P<0.05). Multivariate analyses showed that age older than 52 years and non-CR status at transplant were associated with an increased risk for mortality after RIST.

Conclusions: Our study showed that RIST with this Flu/Bu8 regimen is feasible with durable engraftment and low early mortality. The clinical response rate is adequate for those who stayed in remission at transplantation, but otherwise a novel approach to prevent disease progression should be developed.
Pharmacokinetics and Tolerability of Intravenous Busulfan in Hematopoietic Stem Cell Transplantation.
Authors: Yoon Sung-Soo , Cho Yo-Han , Lim Hyun-Ae , Kim Inho , Bang Soo-Mee , Lee Jong Seok , Park Seonyang , Kim Byoung KooK
Published: October 2019

Intravenous (IV) busulfan has been developed to overcome variable absorption of oral busulfan and tested in several trials. We tested its pharmacological properties and tolerability in 16 Korean SCT patients. IV busulfan was administered at 0.8 mg/kg every 6 hours for a total of 16 doses (days -7 to -4), which was followed by cyclophosphamide administration at 60 mg/kg every 24 hours for 2 days (days -3 and -2). The median AUCinf values (at the 1st dose) and AUCss (at the steady state) were 1060.4 mM·min (range: 511.1– 1812.7) and 1092.5 mM·min (range: 539.7 – 1560.8), respectively. All patients had an AUCinf of < 1500 mM·min at the 1st dose, and 13 of the 16 (81.3 %) maintained AUCss levels between 800 and 1500mM·min. Overall, pharmacokinetics of IV busulfan in our SCT patients appeared comparable to those observed in other study. Thirteen of 16 patients achieved successful engraftments but 4 patients (25%) developed hepatic VOD (2 of which were fatal). Although there was no apparent correlation between AUC and hepatic VOD development, 3 out of 4 patients with VOD had advanced disease at the time of SCT. Notably, patients who had uncontrolled diseases (CML blast crisis, ALL 2nd relapse) succumbed to VOD and those who survived VOD had less advanced disease (CML CP, ALL in 2nd CR) at the time of SCT. In conclusion, pharmacokinetics of IV busulfan in our SCT patients appeared comparable to those observed in other studies, however, hepatic VOD was still a major morbidity, especially in patients with advanced disease, warranting consideration of other parameters in addition to pharmacokinetic monitoring of IV busulfan to decrease VOD in high risk patient populations.

Hepatic veno-occlusive disease cases (n=4) sex/age disease state AUCinf AUCss Outcome Comment F/41 CML blast crisis 511.0 539.7 death unrelated donor F/49 CML chronic phase 739.4 991.0 recovered F/39 ALL 2nd CR 1472.1 1465.6 recovered M/17 ALL 2nd relapse 1302.7 1560.8 death pseudomonas sepsis
Respiratory Syncytial Virus Infection Outbreak and Successful Treatment with IVIG and Oral Ribavirin among Pediatric Patients with Oncologic Diseases and/or BMT.
Authors: Anak Sema S. , Atay Didem , Garipardic Mesut , Ozalp Beril , Yani Diana , Agaaoglu Leyla , Ozturk Gulyuz , Unuvar Aysegul , Karakas Zeynep , Devecioglu Omer
Published: October 2019

Respiratory syncytial virus (RSV) has been reported to cause severe morbidity and mortality among cancer patients receiving chemotherapy together with or without autologous/allogeneic stem cell transplantation (APBSCT), but there have been few reports describing the outcome of RSV infection specifically among pediatric oncology patients. Between February 20 – April 15, 2006, among 15 pediatric patients hospitalized for various oncologic diseases and post-BMT problems, 7 patients developed progressive cough and/or dyspnea. A survey of respiratory viruses was done using direct immunofluorescent antibody assay. Three patients (two high-risk patients with acute lymphoblastic leukemia receiving induction or consolidation chemotherapy, one under treatment for chronic GvHD postBMT) were found positive for RSV (20%). The remaining patients with respiratory symptoms were followed-up for RSV infection, but remained negative during all surveys. Three patients and the sister of the boy with hyper-IgM syndrome, who was also transplanted for hyper-IgM syndrome and under treatment for chronic GvHD and pneumonia, in the same room with her brother, were all treated with IVIG and specific antiviral therapy, oral Ribavirin (20–25 mg/kg/day in three doses). All patients recovered fully, although two were retreated due to recurrent RSV positivity and respiratory symptoms within two weeks. In striking contrast with the outcome of RSV infection in adult oncology patients, the mortality associated with RSV infection in pediatric oncology patients even in postBMT period, is very low and can easily be treated with IVIG and oral Ribavirin, used effectively because of unavailability of other forms (iv or nebulized forms) in our country, if diagnosed and treated early enough. It is even possible to give the scheduled anti-neoplastic therapy for not being delayed.
Analysis T Cell Receptor Excision Circles (TRECs) in Patients with Graft-Versus-Host Disease (GVHD) after Allogeneic Stem Cell Transplantation.
Authors: Du Xin , Li Yangqiu , Weng Jianyu , Lu Zesheng , Guo Rong , Chen Shaohua , Yang Lijian , Geng Suxia , Huang Zhilun
Published: October 2019

The thymic-dependent pathway that involves generation of new naive T cells from donor-derived precursor cells accounts for the more durable reconstitution of the T-cell compartment and generates a more diverse TCR repertoire. Thymic function and production of recent thymic emigrants (RTEs) may be directly evaluated through the quantification, by real-time polymerase chain reaction (PCR), of the T-cell receptor excision circles (TRECs). Following hematopoietic stem cell transplant (HSCT), there is a prolonged period of profound immune deficiency, which continues for years after HSCT. The factors that inhibit thymic function may include age, graft-versus-host disease (GVHD), and direct thymic damage from chemoradiotherapy. GVHD after HSCT also leads to thymic insufficiency, possibly by direct attack on the thymic stroma by allogeneic effector cells. The aim of our study is to analysis T cell receptor excision circles (TRECs) in patients with GVHD after allogeneic stem cell transplantation. We used real-time polymerase chain reaction (PCR) to quantify SjTRECs in 12 patients with GVHD(9 males, 3 females; median age 32 years old), who underwent HLA-matching sibling BMT and/or peripheral blood stem cell transplantation (PBSCT) at our department. Quantitative detection of sjTRECs in DNA of peripheral blood mononuclear cells from 13 normal individualals. The median value of sjTRECs copies P1 000 PBMCs was 4.37±3.64 in normal indiviuals. However, the decreased levels of TRECs were most profound in the group of patients with active chronic GVHD at the time of study. it was 0.26±0.22 copies P1 000 PBMCs in 12 patients with GVHD (P < 0. 00007). We conclude that measurement of sjTREC may provide an important tool for predicting thymus-dependent T-cell reconstitution in GVHD patients after transplantation.
Puvatherapy in the Management of Graft-Versus-Host Disease: Brazilian Experience.
Authors: Bouzas Luis Fernando , Silva Marcia M. , Lerner Decio , de Cassia B. Tavares Rita , Azevedo Alexandre M. , Filgueira Absalom L.
Published: October 2019

Background: Graft-versus-host disease (GVHD) is a frequent late complication of allogeneic hematopoietic stem cell transplantation(HSCT) and remains a difficult problem to treat and manage. Therapy with 8-methoxypsoralen (8-MOP) and ultraviolet A to control drug resistant chronic graft-versus-host disease(c-GVHD) was based on the induction of functional changes in the effectors of the disease (T lymphocytes and cytokines). The potentially beneficial effect of phototherapy for treatment of cutaneous manifestations of GVHD led us to investigate retrospectively the effect of this therapy in a larger series of patients.

Methods: From May 1991 to April 2005, 43 patients post-bone marrow transplanted for malignant and non-malignant diseases, with c-GVHD were treated with PUVA. Extensive c-GVHD was diagnosed in 39 patients and 4 patients had limited disease. PUVA was administered three times a week with no oral mucosa direct exposure. 8-MOP dose was 0.6mg/Kg given two hours before UVA irradiation and initial dose was based on skin type(Fitzpatrick).

Results: From forty-three patients with resistant chronic graft-versus-host disease who were submitted to PUVAtherapy, three patients had began recently the treatment (less than 20 sessions) and were considered early to evaluated. Seven could not be evaluated because they had abandoned the treatment before complete 20 sessions. Early side effects were: nauseas, vomiting and gastric intolerance in 4 patients, burning in 6, photophobia in 2 and chronic effects were lentigo-PUVA in 1, guttate leukoderma in 1 and basal cell carcinoma in 1 patient. Immunosuppressive therapy was reduced in 7 patients and discontinued in 18 post-PUVAtherapy. Overall 25 patients(75,6%) responded to PUVA. Fifteen patients had complete response and 10 a partial response during PUVA treatment. Six patients(54,5%) from eleven had died from GVHD progression, during PUVAtherapy. The Kaplan-Meier estimate of survival at 3 years after the start of PUVAtherapy was 76%.

Conclusion: PUVAtherapy was effective for tegumental c-GVHD resistant to immunosuppression, improving the quality of life of these patients. Evidence of systemic effect of PUVAtherapy could be observed by oral lesions healed in all patients without direct exposure.
Predicting Risk of Infection and Graft vs. Host Disease (GvHD), and Response to Immunosuppression (ISP) in Allogeneic Hematopoietic Stem Cell Transplant (HSCT) Patients (pts) Using the Cylex®ImmuKnow® Assay: A Pilot Study.
Authors: Anaissie Elias J. , Diaz Jose A. , Grazziutti Monica L. , Miceli Marisa H. , Fox-Cottler Michele , Barlogie Bart
Published: October 2019

Background: GvHD, intensity of ISP and infection are major causes of toxicity post-HSCT. Yet, no method reliably predicts these complications. A functional assay for immunity, which predicts risk of GvHD and infection and allows tailoring ISP and infection prophylaxis, is needed. The ImmuKnow is an assay of global T-cell function that measures increased ATP production after stimulating CD4+ T-helper cells and has been shown useful for managing ISP therapies in solid-organ transplantation. We hypothesized that ImmuKnow could assist in predicting risk of GvHD and infection and response to ISP post-HSCT.

Patients and Methods: Between January 2003 and May 2006, 34 adult allogeneic HSCT pts [mean age 47 years (range 22–71); 53% females; acute myelogenous leukemia, 8 pts; myeloma, 6 pts;] underwent blood sampling for determining ImmuKnow at various times post-HSCT (238 assays, mean of 7/pt). Results were correlated with clinical course (infection, stable, GvHD, response to ISP).

Results: During the testing period, GvHD was documented in 16 pts [(acute, n=7 pts; chronic, n=9 pts] and 53 infections occurred [CMV viremia (n=20); bacteremia (n=20); others (n=13)]. The median immune function (ng/mL ATP) of GvHD pts was significantly higher than that of clinically stable pts (342 +/−105 vs. 167 ng/mL +/−136 respectively), p<0.001. Pts with infections had a median 89 ng/mL ATP (+/−119), also statistically different (p=0.002) from stable pts. A pt with ATP value ≥ 300 ng/mL was more likely (RR=4) to develop GvHD than one with lower immune response. Similarly, there was a higher risk (RR=5) for infection in pts with an ATP ≤ 80 ng/mL. Relative risk curves for infection and GvHD intersected in the low immune response zone (140 ng/mL ATP). Following therapy for GvHD in 10 pts, ATP values rapidly decreased (193 ng/mL ATP +/−122) over 17 days (range 3–35) and were associated with improvement, while stable or increasing values (50 ng/mL ATP +/−99) were observed among 6 pts who failed to respond to GvHD therapy.

Conclusions: these data suggest that the FDA-approved ImmuKnow assay is an objective marker of risk for infection and GvHD, and of responsiveness to ISP therapy post-HSCT. Tailoring intensity and duration of ISP to risks of GvHD and infection in an individual pt may be possible and may improve outcomes. Validation of these preliminary data is ongoing.
Safety and Efficacy of Cytotoxic Chemotherapy after Allogeneic Hematopoietic Stem Cell Transplantation for Relapsed Hematologic Malignancy.
Authors: Hardy Nancy M. , Odom Jeanne , Snow Kelly , Dean Robert , Pavletic Steven , Sportes Claude , Gress Ronald , Fowler Daniel H. , Bishop Michael R.
Published: October 2019

Relapse of hematologic malignancy is a major problem after allogeneic hematopoietic stem cell transplantation (alloHSCT), with limited treatment options of proven benefit other than donor lymphocyte infusions (DLI). Additionally, toxicity from treatment with cytotoxic chemotherapy after alloHSCT may differ from conventional treatments, and could include graft-versus-host disease, allograft failure, or increased risk of infection. We retrospectively reviewed treatment outcomes of 25 patients who received cytotoxic chemotherapy as treatment for relapsed hematologic malignancies after reduced-intensity, matched-sibling donor alloHSCT. Combination therapies included: EPOCH+/− rituximab (n=12 patients); “7&3” AML induction (ida/ara-c) (n=2); FLAG (n=2); asparaginase/6-MP/vincristine (n=1); R-ICE (n=1); MIME (n=1); BVP (n=1); and bortezomib/doxorubicin/dexamethasone (n=1). Single-agent therapies included: bortezomib (n=6); gemcitabine (n=4), vinorelbine (n=3); vincristine (n=1); methotrexate (n=1); and intrathecal methotrexate and/or cytarabine (n=5). A total of 133 cycles or doses were administered, of which 20 were supported with donor stem cell boosts, and another 9 were followed by nonmobilized DLI. There were 52 Grade 3 or higher toxicity episodes recorded, most commonly: neutropenia (10 episodes); infection with neutropenia (5 episodes); thrombocytopenia (6 episodes); and anemia (3 episodes). There were 12 episodes of CMV reactivation in 8 of 15 patients at risk, including 2 cases of pneumonitis and 1 case of colitis. GVHD flares were considered definitely chemotherapy-induced (2), possibly chemotherapy-induced (7), and unlikely chemotherapy-induced (2). Unusual events after treatment included culture-negative sepsis-like illness after bortezomib (n=2), secondary (11q23) AML of donor origin after one cycle of EPOCH (n=1) and diffuse alveolar hemorrhage after EPOCH (n=1). Seven patients achieved CR with therapy for post-transplant relapse, including three durable remissions. Four patients achieved PR and seven achieved disease stabilization. Median survival after starting cytotoxic therapy was 263 days. Timing of relapse appeared to be associated with survival, with median overall survival of 95 days for patients requiring therapy before Day 100 vs. 508 days for patients after Day 100 (p=0.0008). Treatment of relapse with cytotoxic chemotherapy after alloSCT is feasible and can result in durable remissions in a minority of patients. However, administration of cytotoxic therapy after alloHSCT requires monitoring and support for hematologic toxicities, GVHD and CMV reactivation; selected patients may benefit from prophylactic stem cell boosts or immune suppression.

Survival after Cytotoxic Therapy for Relapse after alloHSCT Survival after Cytotoxic Therapy for Relapse after alloHSCT
Compared Molecular Monitoring of BCR-ABL Transcripts in Peripheral Blood and Bone Marrow Samples in CML Patients after Allogeneic Hematopoiteic Stem Cell Transplant.
Authors: Dominietto Alida , Cirmena Gabriella , Garuti Anna , Raiola Anna Maria , Ibatici Adalberto , van Lint Maria Teresa , Frassoni Francesco , Bacigalupo Andrea
Published: October 2019

Background. Molecular monitoring of the BCR-ABL transcripts in patients with chronic myeloid leukemia (CML) using quantitative real-time polymerase chain reaction (QRT-PCR) provides important information about the leukemia cell mass and the response to therapy. After allogeneic hematopoietic stem cell transplants (HSCT) patients with persistently positive levels of BCR-ABL transcript have a molecular relapse and some of these patients progress to develop a cytogenetic or hematologic relapse.

Objectives. To test whether molecular detection of BCR-ABL transcripts is comparable using peripheral blood (PB) and bone marrow (BM) aspirate samples after allogeneic HSCT.

Patients and Methods. BCR-ABL transcripts were monitored the interval of time 2003 – 2005 in 118 patients who received an allogeneic HSCT in chronic or advanced disease phase. BCR-ABL transcripts were evaluated concomitantly in 200 blood samples and 200 marrow samples (total 400 determinations) using real time PCR (QRT-PCR) assay to analyze BCR-ABL gene rearrangement (p210 b2a2 and b3a2). A complete molecular response (CMR) both for PB and BM we defined has undetectable levels of p210 and Major molecular response (MMR) was defined when p210/ABL ratios where <0.02%.

Results. 135 double BM+PB determinations (67%) proved BCR-ABL negative; 61 double determinations (31%) were classified as “persistently positive” both in peripheral blood and in blood marrow; 4 determinations (2%) were discordant. Therefore 196/200 BCR-ABL determinations in peripheral blood and bone marrow (98 %) were concordant.

Conclusions. This study shows that BCR-ABL QRT PCR monitoring of CML patients after allogeneic HSCT with peripheral blood cells is concordant with bone marrow cells in 98% of cases, and thus may be used to monitor the disease. This may be relevant for patients, especially when quality of life issues are discussed together with the need for post-transplant monitoring.
A Prospective, Randomised Trial of Chlorambucil, Mitoxantrone and Dexamethasone (CMD) Versus Fludarabine, Mitoxantrone and Dexamethasone (FMD) for Advanced Follicular Lymphoma (Real Grades I-III, Stages III/IV).
Authors: Haynes Andrew , McMillan Andrew , Jack Andrew , Qian Wendi , Jakupovic Ira , Smith Paul , Prentice Archie
Published: October 2019

Fludarabine, alone and in combination, has activity in de novo and relapsed/refractory follicle centre cell lymphoma (FCCL), as do many other combinations of chemotherapy, including chlorambucil, mitoxantrone and steroids. In this multicentre study, between May 2000 and April 2006, 400 adults with FCCL (REAL grades I-III) were randomised to receive either CMD (chlorambucil 10mgs po, od, days 1–10, mitoxantrone 12mgs/m2 IV, day 1 and dexamethasone 20mgs po, od, days 1–5) or FMD (fludarabine 25mgs/m2, IV days 1–3, with M and D as for CMD) on physicians’ decision to treat advanced stage (III or IV) disease (B symptoms, bone marrow failure, bulk or progression and compression syndromes). Rituximab (R) was not available for this trial. Randomisation was prospectively stratified by IPI score. Patients received four courses before assessment of response, then had no further trial therapy with progression or inadequate response or had a maximum total of eight courses if responding. The primary end points were progression-free survival (PFS) and overall survival (OS) and the secondary end points were complete (C) and partial (P) remission rates (RR). This preliminary report is at median follow-up of 31 months (range 0–71). Clinical features and pathological diagnoses (reviewed centrally) were equivalent across both arms. The median age in years (range; number >60 y) for CMD was 56 (32–72; 68) and for FMD 55 (29–75; 68) and 70% in both arms had stage IV FCCL. For CMD versus FMD, the CRR and PRR after course 4 were 18% v 21% and 72% v 68% and after course 8 or at the end of treatment were 44% v 45% and 47% v 47%. The hazard ratios were in favour of CMD, for OS at 1.65 (95%CI 0.97–2.8, p=0.063) and for PFS at 1.61 (95%CI 1.2–2.17; p=0.0018). The numbers of deaths due to lymphoma were equivalent (19 v 21) but there were fewer deaths in the first year (most due to lymphoma in both arms) with CMD (7 v 15; ns). The difference between those who received between 6 and 8 courses in either arm was not significant (152[80%] v 135[71%]). With CMD there were higher rates of grade 3/4 neutropenia (8 v 1) and infections (29 v 20) but these differences were not statistically significant; other toxicities were low grade and equivalent for both arms. Days between cycles (median 28 days in both arms) and dose adherence (96% and 97%) were equivalent; both regimens were well tolerated and relatively easy to deliver. The equivalent dose intensity, toxicities and RRs of the two arms indicate that the statistically significant superior PFS for CMD was not due to undertreatment in the FMD arm. The outcomes for CMD are at least as good as those reported for other combinations of chemotherapy without R and R-CVP.
Fludarabin/Cyclophosphamide Reduced Intensity Conditioning for Allografting Offers an Excellent Outcome for Heavily Pretreated Lymphoma Patients.
Authors: Jonkhoff A. Roel , Janssen J.J.W.M , Visser O. , van Roessel H.M. , Huijgens P.C.
Published: October 2019

To test the value of non-TBI based reduced intensity stem cell transplantation (RIST) in high risk patients with NHL, CLL and MM, 52 patients were transplanted using fludarabin 25 mg/m2 and cyclophosphamide 500 mg/m2, for 5 days and an undepleted HLA-identical sibling allograft.

Patients: diagnosis: indolent NHL (10), diffuse large cell lymphoma (8), mantle cell lymphoma (2), multiple myeloma (17), CLL (10), other diagnosis (5).

Patient characteristics: 34M/18F, median age 52.9 yr (range 18–66 yr). All patients were extensively pre-treated. None of CLL patients had received an autologous transplant, but of all remaining patients 71% had been autografted previously. Best response in NHL patients at transplant: CR in 2; PR in 16; PD in 2. The graft consisted of 6.8 × 106/kg CD34+ cells (median, range: 2.7 – 20) and T-cells 34.1 × 107/kg (range 14.5–66).

Results: Hematological recovery: nadirs respectively: WBC: 0.34 × 109/l (median, range <0.1 – 1.6) at day +4, platelets: 38 × 109/l (2–130) at day 8, ANC: 0.09 (0 – 0.36). Toxicity was minimal: mucositis > grade I: 4 patients; TPN: 7 patients; antibiotics i.v.: 8 patients; hemorrhagic cystitis: 2.

Engraftment was prompt in all but one patient. Chimerism measured by short tandem repeats (STR) at day +30, +100 and +180 were 85%, 95% and 95% respectively (median, range 15 – 100%). Acute GVHD grade I/II was seen in 10 (19%) and grade III/IV in 11 (21%). Chronic GVHD occurred in 12 patients (limited 5 (10%), extensive 7 (13%). Transplant related mortality was only 10%. With a median follow-up of 48 months for NHL patients the estimated survival is 78%. For CLL and MM patients follow-up is still to short for conclusions, but survival seems inferior in MM patients

Conclusion: fludarabin/cyclophosphamide allogeneic transplantation provides an excellent rescue treatment for heavily pretreated lymphoma patients.

Survival Flu/Cy allogeneic RIST transplantation Survival Flu/Cy allogeneic RIST transplantation
A Fludarabine-Based Non-Myeloablative Conditioning Regimen in Allogeneic Stem Cell Transplantation from Related and Unrelated Donors in Chronic Myeloid Leukemia.
Authors: Luo Yi , Huang He , Cai Zhen , Tan Yamin , Han Xiaoyan
Published: October 2019

Objective: To evaluate the efficacy and safety of a fludarabine-based non-myeloablative conditioning regimen in allogeneic stem cell transplantation (SCT)from related and unrelated donor for chronic myeloid leukemia in chronic phase(CML-CP).

Methods: Fifteen consecutive patients with CML-CP between May, 2005 and July, 2006 were treated with a single non-myeloablative conditioning regimen in this study. They were 10 males and 5 females with a median age of 41 years (range, 18–49). Donors were HLA-A, B and high resolution DR fully matched siblings (n=8), matched unrelated donors (n=6), and 1-locus mismatched unrelated donors (n=1). The stem cells were collected from either peripheral blood (n=9) or bone marrow (n=6). The conditioning regimen included fludarabine 30 mg/m2/day (days -10 to -5), oral busulfan 4 mg/kg/day (n=4 patients), or intravenous busulfan 3.2 mg/kg/day (n=11 patients) (days -6 to -5) and anti-thymocyte globulin (Fresenius, Germany) (5mg/kg/day) (days -4 to-1). Mycophenolate mofetil combined with cyclosporin A and methotrexate was used for prevention of acute graft-versus-host disease(GVHD) after transplantation. Lipoprostagandin E1 was used in prophylactic regimen for hepatic veno-occlusive disease(VOD). To assess engraftment, degree of chimerism, minimal residual disease and relapse, all patients were monitored by cytogenetic analysis and donor vs host-specific DNA markers using short tandem repeats (STR) assay. The average cell number of MNC transfused was 4.83 (3.14~11.5)×108/kg; CD34+ cells were 3.47(2.38~6.24)×106/kg, CFU-GM was 2.15 (1.85~3.06) ×105/kg.

Results: Engraftment of neutrophils and platelets was achieved in 14 out of 15 (93.3%) patients within a median of 13 days (range, 8–21) and 18 days (range, 10–35), respectively. Fourteen patients achieved complete donor chimerism in the peripheral blood before day +35 and one developed graft failure. No patients developed acute GVHD and VOD, but one died from interstitial pneumonia while she was in continuous complete remission 2 months following transplantation. With a median follow-up of 5 months (range 1.5 to 15), 13 of them were still in CCR. The overall non-relapse mortality in this group was 6.67% (1/15 patients). Overall survival, and disease-free survival rates were 93.3% and 86.7%, respectively.

Conclusion: A fludarabine-based non-myeloablative conditioning regimen in allogeneic stem cell transplantation from related and unrelated donors is an effective and safe choice for patients with chronic myeloid leukemia in chronic phase.
Disappearance of Minimal Residual DEK-CAN Fusion Transcript after Allogenic Hematopoietic Stem Cell Transplantation in a Child with t(6;9) Acute Myeloid Leukemia.
Authors: Kawasaki Hirohide , Imai Yuichiro , Kuroyanagi Yuichi , Noda Yukihiro , Nakano Takahide , Kaneko Kazunari
Published: October 2019

Chromosomal translocation t(6;9) (p23;q34) associates with acute myeloid leukemia (AML), although that is found in less than 2% of all cytogenetic events in children with AML. Recent molecular analysis of patients with t(6;9) (p23;q34) has demonstrated a rearrangement of the genes DEK and CAN resulting in the formation of a chimeric DEK-CAN mRNA. The prognosis of patients with t(6;9) is thought tobe very poor, because of poor response to chemotherapy and/or high relapse rate. However, recent reports showed that allogeneic hematopoietic stem cell transplantation may have a potential to improve the prognosis of patients with t(6;9). We report here we were able to monitor DEK-CAN fusion transcripts in the serial points along chemotherapy courses and hematopoietic stem cell transplantation in a child with t(6;9). A 5-yaer-old Japanese boy diagnosed as having AML (M5b). Chromosomal analysis revealed t(6;9)(p23:q34), and real-time quantitative polymerase chain reaction (RQ-PCR) revealed positive chimeric DEK-CAN mRNA. After diagnosis, he received an induction chemotherapy. At the end of the induction chemotherapy, he was in a hematological and cytogenetic complete remission (CR), however DEK-CAN fusion transcript was still positive. After completion of five courses of consolidation therapy, he was in hematological and cytogenetic CR, although molecular minimal residual disease (MRD) was still positive. So he received allogeneic peripheral blood stem cell (PBSC) transplantation from 1 locus mismatched father. Busulfan and Melpharan were used for conditioning regimen. Cyclosoprin A and short course of methotrexate were emplyed for the prophylaxis of graft-versus-host disease (GVHD). Engraftment of PBSC was achieved at day 15. He had mild acute GVHD and no clonic GVHD. Bone marrow analysis at the time of engraftment revealed hematological, cytogenetic, and molecular CR. Now he is still in a persistent molecular negativity of DEK-CAN fusion transcript by RQ-PCR and keeps event-free survival for five years after PBSCT. The monitoring of DEK-CAN fusion transcript in the chemotherapies was reported from some investigators. However, there were very few reports about the monitoring DEK-CAN fusion transcripts before and after hematopoietic stem cell transplantations. In coclusion, we succeeded to exclude DEK-CAN fusioin transcript after hematopoietic stem cell transplantation in a child with t(6;9) AML. We conclude that DEK-CAN molecular monitoring by RQ-PCR in is a useful tool for the management of patients with t(6;9) AML.
Haploidentical Stem Cell Transplantation without T Cell Depletion: A Brazilian Protocol.
Authors: Simoes Belinda P. , Navarro George , Favarin Maria C. , Palma Leonardo C. , Ayrosa Maria I. , Velano Carlos E. , Pieroni Fabiano , Stracieri Ana Beatriz P.L. , Moraes Daniela , Oliveira Maria C. , Voltarelli Julio C.
Published: October 2019

For patients with refractory or high risk leukemias, stem cell transplantation is still the only possible curative approach. Those patients, although, without a HLA identical matched sibling, unrelated stem cells are the only stem cell source. In patients with refractory disease this unrelated cells may be unavailable in a short time period. The use of HLA-mismatched familiy donors has been used before, by depleting in vitro T cells or selecting CD34+ cells. These methods are extremely expensive and patients suffer with delayed immune reconstitution. In an attempt to overcome the HLA barrier, Cyclophosphamide, a highly toxic agent against activated T cells, but not myeloablative, has been used after transplantation to ensure engraftment and avoid GVHD. We describe here two patients who were sequentially submitted to a citoreductive protocol described by Schmid et al (Blood 2006) followed after transplantation by Cyclophosphamide (50 mg/kg day +3 and day +4) as described by O’Donnel et al (BBMT 2002). We transplanted 2 boys (16 years-case 1 and 6 years-case 2) with secondary AML after MDS and AML relapse after second SCT respectively. Case 2 received two SCT from his HLA-id brother and was 8 months after the second transplant. Both had active disease at time of transplant and received full bone marrow from there HLA haploidentical mothers (3 loci mismatch). Engraftment was achieved on day +15 in both cases. Chimerism analysis on day +30 showed that they had 96% XX and 95% XX cells respectively in bone marrow. Case 1 never developed GVHD but relapsed after SCT. Case 2 are in complete remission and complete quimera day+90 after SCT. He developed GVHD (skin, liver and gut) were treated with prednisone and Basiliximab and we are now tapering imunossupression.

Herby we demonstrated that haploidentical transplantation with the use of Cyclophosphamide after transplantation can be a useful transplant strategy in high risk patients without a matched sibling donor. Relapse and infections will always be the most frequent complications. In countries with limited health resources a protocol who avoid the expensive costs of cell depletion or selection can be a real alternative for patients without suitable donors.
The B4galnt2 Regulatory Polymorphism, Mvwf1, Causes Low VWF Levels and Segregates in Natural Mouse Populations
Authors: Johnsen Jill , Baines John , Tautz Diethard , Ginsburg David
Published: October 2019

We previously identified the cause of low levels of von Willebrand factor (VWF) in the RIIIS/J mouse strain to be a regulatory mutation, Mvwf1, in an N-acetylgalactosaminyltransferase, B4galnt2 (previously designated Galgt2). Mvwf1 causes a tissue-specific switch in B4galnt2 expression from intestinal epithelium to vascular endothelium, resulting in aberrant glycosylation of VWF and accelerated clearance from circulation. We have identified thirteen Mvwf1 inbred mouse strains that share this remarkable tissue-specific switch and a common 97kb haplotype block, including a 30kb region of 2–3% sequence divergence that flanks Exon 1. An RIIIS/J BAC transgene containing the entire Mvwf1 haplotype block and B4galnt2 gene confers vascular gene expression, while C57BL6/J BAC transgenes spanning the homologous region confer “wild-type” vessel(−), intestine(+) gene expression, indicating that one or more tissue-specific regulatory elements sufficient to recapitulate the Mvwf1 tissue-specific switch lie within the genomic region covered by these BACs. A wild-derived recombinant Mvwf1 allele containing the 3′ half of the Mvwf1 haplotype block confers the vessel(−), intestine(+) B4galnt2 expression pattern, placing the regulatory mutation(s) responsible for the Mvwf1 switch well upstream of the proximal promoter region. Sequence analysis of DNA from wild-caught individual mice confirmed the presence of a highly conserved wild mouse Mvwf1 founder allele that likely pre-dates the development of the inbred mouse strains. PCR of genomic DNA from wild caught mice representing M. m. musculus, M. m. domesticus, M. m. castaneus, M. m. molossinus, M. spretus, M. hortulanis, and M. macedonicus revealed that the Mvwf1 allele is common in wild M. m. domesticus populations in North America, Europe, and Africa, with an allele frequency as high as 60% in French mice. Population samples of M. m. domesticus from Cologne, Germany and the Massif Central region of France drastically differ in the frequency of this allele (0% vs. 60%). A significant reduction in microsatellite variability at B4galnt2 in the French population, as measured by the lnRH statistic, suggests a recent, local shift in the frequency of Mvwf1, likely due to a recent change in selective pressure. Analysis of independently trapped wild mice from the Massif Central region of France confirmed that Mvwf1 confers vascular endothelial B4galnt2 expression and causes significantly lower VWF levels (5.5+2.0 vs. 13.3+2.3, p<1x10−7) in this population. These data support a B4Galnt2 allele-specific survival advantage in wild mice, leading us to speculate that a similar survival advantage could account for the high prevalence of VWD in human populations.
Single Versus Multiple Transplants for Multiple Myeloma: Insights from In Vivo and In Silico Studies.
Authors: Dingli David , Pacheco Jorge M. , Gertz Morie Abraham
Published: October 2019

Background: The advent of high-dose therapy and autologous stem cell transplantation (HDT-ASCT) has significantly improved response rates and survival for patients with multiple myeloma (MM). However this form of therapy is potentially toxic and does not cure patients from their disease. The response to HDT-ASCT is variable and approximately 40% of patients achieve a complete response (CR). Patients with a CR have a significantly improved time to progression (TTP) compared to patients who do not achieve CR (nCR). Optimal timing of HDT-ASCT and considerations of the relapse kinetics may assist treatment planning for this disease. We analyzed myeloma tumor burden and kinetics before and after HDT-ASCT in order to define optimal timing of this therapy for the individual patient.

Patients and Methods: A cohort of 265 patients with MM who underwent HDT-ASCT at Mayo Clinic Rochester was studied. Disease burden before and after HDT-ASCT was estimated from serial M-spike measurements. The estimated tumor burden data were fitted to the Gompertz function and its parameters alpha and beta estimated using a non-linear least squares method. Cohorts of patients with CR and nCR were studied separately. Functions that coupled disease burden and kinetics with TTP were derived and used to determine the optimal timing of single and tandem transplantation.

Results: The Gompertz parameters alpha and beta for patients who achieved CR and for those who did not achieve CR with HDT were determined. Our values were lower than those of Sullivan and Salmon although the ratio of alpha/beta was of a similar order (implying the same carrying capacity). Using the idealized Gompertz parameters, simulations of single versus tandem transplants were performed. Our model predicts that tandem transplantation increases CR rate by approximately 10%. When the time between transplants was studied as a variable, our model suggests that the optimal time between transplants is 7 months.

Conclusions: Our modeling provides a theoretical understanding of tumor burden and kinetics for patients with MM who undergo HDT-ASCT. Patients who achieve CR with the first episode of HDT-ASCT should not be routinely offered tandem transplantation but carefully monitored and re-transplanted at an optimal disease burden. If CR is not achieved with a first cycle of HDT-ASCT, the probability of CR after a second trial of HDT is ~10% (similar to that reported by Attal et al 2003) although this depends on the tumor burden before the second transplant. TTP after tandem transplants (with its higher associated mortality) cannot be superior to TTP achieved with optimally timed serial transplants. Individualized HDT-ASCT for patients with MM is possible and may improve results.
High Dose Therapy and Autologous Hematopoietic Cell Transplantation for Primary Refractory Diffuse Large Cell Non-Hodgkin’s Lymphoma.
Authors: Cao Thai M. , Stockerl-Goldstein Keith E. , Cao Paul D. , Laport Ginna G. , Sheehan Kevin , Shizuru Judith A. , Johnston Laura J. , Negrin Robert S. , Lowsky Robert
Published: October 2019

The efficacy of high dose therapy (HDT) and autologous hematopoietic cell transplantation (AHCT) for patients with diffuse large cell non-Hodgkin’s lymphoma (NHL) who never achieve a first complete remission (CR) with conventional chemotherapy have been addressed by few published studies. We retrospectively analyzed outcomes for 43 consecutive patients with chemotherapy-sensitive primary refractory diffuse large cell NHL treated with HDT and AHCT at our center between November 1988 and January 2002. The median age at transplant was 43 years (range: 18 – 64). At diagnosis 26 patients (60%) had stage IV disease, 28 patients (65%) had extranodal involvement, and bulky disease was present in 17 patients (40%). The age-adjusted NHL international prognostic index (IPI) score was low in 4 patients (9%), low-intermediate in 18 patients (42%), high or high-intermediate in 12 patients (28%), and not available in 9 patients (21%). All patients failed to achieve a first CR following CHOP (n = 31, 72%), R-CHOP (n = 6, 14%), and other anthracycline-containing regimens (n = 6, 14%). Fourteen patients (33%) did not receive additional conventional chemotherapy before proceeding to HDT and AHCT. The remaining 29 patients (67%) received in addition between 1 – 6 cycles of salvage chemotherapy. None of the 43 patients had achieved a CR at any time point before HDT and AHCT and all patients had measurable disease at the start of the HDT regimen. The median interval from diagnosis to HDT was 8.9 months (range: 2.4 – 15.5). The HDT regimen consisted of total body irradiation, etoposide, and cyclophosphamide in 15 patients (35%); BCNU, etoposide, and cyclophosphamide in 26 patients (60%); and CCNU, etoposide, and cyclophosphamide in 2 patients (5%). All but three patients (7%) were rescued with peripheral blood stem cells mobilized with cyclophosphamide 4 gm/m2 plus G-CSF and purged with monoclonal antibodies and complement. With a median follow-up of 5.8 years (range: 0.2 – 15.8) among surviving patients, the 5-year Kaplan-Meier estimates for overall survival was 67.4% (95% CI: 52.4 – 82.4), freedom from progression was 60.1% (95% CI: 43.7 – 76.6), and event-free survival was 55.8% (95% CI: 39.2 – 72.4). Two patients with relapse post-AHCT proceeded to non-myeloablative allogeneic HCT and were censored at the time of second transplant. By univariate analyses the following characteristics were not prognostically significant for overall survival: disease stage at diagnosis (I–II versus III–IV), prior radiotherapy, age-adjusted IPI score (low versus high) in evaluable patients, and the HDT regimen (TBI versus non-TBI). These results demonstrate that HDT and AHCT is effective treatment for chemotherapy-sensitive primary refractory diffuse large cell NHL.

Figure Figure
Efficient Viral Transduction of Murine Hematopoietic Stem Cells Using HIV-1 Gag-Pol Cross Packaged Simian Immunodeficiency Viral Backbone.
Authors: Lin Yuan , Gerson Stanton L.
Published: October 2019

Lentiviral vectors have been shown to infect non-dividing cells, including hematopoietic stem cell [HSC], and HIV lentiviral vector has been studied extensively in preclinical models. However low HIV lentiviral vector transduction efficiency compared to retroviral vectors, is seen in murine HSC, hampering transplantation and long-term expression of transgene in the recipients. Furthermore, concerns remain regarding the safety of HIV based vectors. Simian Immunodeficiency Viral [SIV] vectors could be safer since the parent virus does not cause disease in humans. However, to model this approach has been difficult because native SIV vectors do not transduce murine cells. We have generated a bicistronic SIV lentiviral SIN vector, containing MGMT and firefly luciferase genes linked by a self-cleavage FMDV 2A sequence. The SIV backbone was kindly provided by Dr. Donald Kohn (University of Southern California). The transgenes are controlled by the MND promoter, which has been shown to express well in murine hematopoietic stem cells. The vector was generated by cross-packaging SIV RNA with HIV-1 ΔR8.91 packaging plasmid and VSVG pseudotyped envelope (Ref. Retrovirology2005, 2:55). Unconcentrated viruses had an average titer of 1E+06 iu/ml, which was similar to HIV-1 lentiviral vectors. In vitro, HIV-1 cross-packaged SIV-mnd-MGMT-2A-Luc vector was able to transduce both human and murine cell lines with no reduction of expression for 10 weeks. In addition, this cross-packaged SIV vector was also able to transduce primary murine bone marrow cells from Balb/C mice with low MOI of 0.5 to 1. Transduced primary murine bone marrow cells maintained transgene expression during a 4 week culture. To analyze in vivo expression, Balb/C bone marrow cells were transduced for 48 hrs in cytokines with the HIV-1 packaged SIV vector and transplanted into irradiated recipients. We used bioluminescent imaging (BLI) to monitor the transgene expression and the dynamic engraftment of transduced murine bone marrow cells. At MOI of 0.5 or 5, transduction efficiencies in murine progenitor cells were 24.4% and 46.7% respectively by PCR of transgene from CFU colonies. Bioluminescent imaging indicated similar engraftment patterns of transduced bone marrow cells by HIV-1 lentiviral vector or cross-packaged SIV lentiviral vector, as early as day 5. Consistent BLI signals indicated sustained expression of transgene in SIV vector transduced bone marrow cells beyond 30 days. With this study, cross-packaged SIV SIN vector could be used as a potential gene transfer vector in both preclinical murine studies and perhaps in clinical trials.