bluuebunny/crossref_metadata_2025 huggingface dataset:
A Comparison of Beam and Yttrium 90 Ibritumomab Tiuxetan (Zevalin®) in Addition to Beam (Z-BEAM) in Older Patients Undergoing Autologous Stem Cell Transplant (ASCT) for B-Cell Lymphomas: Impact of Radioimmunotherapy on Transplant Outcomes.
Authors: Krishnan Amrita Y. , Nademanee Auayporn , Raubitschek Andrew , Fung Henry C. , Molina Arturo , Yamauchi David , Falk Peter M. , Spielberger Ricardo , Palmer Joycelynne , Tsai Ni Chun , Schriber Jeffrey , Forman Stephen J.
Published: October 2019

Background: ASCT is an effective treatment for patients with high risk NHL. However, relapse rates remain high. Attempts at further dose intensification are limited by regimen-related toxicity especially in older patients. Zevalin as a single agent has showed activity in aggressive Non-Hodgkin’s Lymphoma (NHL). We therefore, devised a novel conditioning regimen (Z-BEAM) combining standard dose Zevalin (0.4mci/kg) with high dose BEAM (BCNU 150mg/m2 day-7,-6, cytarabine 800mg/m2 and etoposide 800mg/m2 day-5 to day-2, and melphalan 140mg/m2 day-1).

Herein, we provide an update on the study and also retrospectively compare this new conditioning regimen with conventional high dose BEAM in older patients (age >=50). Between 1995 and 2005, 65 patients (BEAM n=32, Z-BEAM n=33) underwent ASCT utilizing these regimens. Patient selection was similar for Z-BEAM and BEAM. Patients received BEAM prior to the opening of the Z-BEAM protocol and similiarly after the Z-BEAM protocol completed accrual. The groups were well matched for most demographics and disease status. Median age: Z-BEAM - 62 yrs (range 50–78), BEAM - 64 yrs (range 50–77); 1st CR/PR pts comprised 45% in the Z-BEAM arm and 34% in the BEAM, > =1st Relapse 42% vs 50% and induction failure 12% vs 15%. (p=0.7). Median number of prior regimens was 3 in the Z-BEAM group vs 2 in the BEAM arm.(p=0.08) 54 % had bulky disease at diagnosis in the Z-BEAM group vs 41% in the BEAM group (p=0.30).

Results: Two-year OS was 78% for all pts (95%CI, 67–86). Two-year OS/PFS in the Z-BEAM group was 88%(95% CI, 70–95)/72 %(95% CI, 57–83) respectively vs 65% (95%CI, 48–77)/ 67%( 95CI, 50–79) in the BEAM group (Figure1). Analysis by histology showed that in patients with diffuse large B-cell lymphoma (DLBCL), there was a significant difference in OS/PFS of Z-BEAM vs BEAM, 95% vs 48% at 2 yrs (p=.009)/ 88% vs 48% ( p=0.015). The OS/PFS difference of Z-BEAM vs BEAM in patients with mantle cell lymphoma did not reach significance. The toxicity profile and transplant related mortality was similar in both regimens.

Conclusions: Our study suggests that the addition of Zevalin to the BEAM conditioning regimen is feasible and well tolerated in older patients. The favorable outcome of patients treated with Z-BEAM compared with BEAM alone, especially in patients with DLBCL, suggests that prospective study in a randomized trial is warranted.

Overall Survival   ZBEAM versus BEAM Overall Survival   ZBEAM versus BEAM
Autologous Stem-Cell Transplant with a Rituximab Purge and Maintenance vs. Standard Chemotherapy for Mantle Cell Lymphoma: Extended Follow-Up of a Matched Pair Analysis.
Authors: Hicks Lisa , Connors Joseph M. , Mangel Joy , Buckstein Rena , Crump Michael , Leitch Heather , Imrie Kevin , Nagy Tracy , Boudreau Angela , Pennell Nancy , Spaner David , Berinstein Neil
Published: October 2019

Background: We previously reported that 20 patients with newly diagnosed, stage III/IV mantle cell lymphoma (MCL) treated prospectively with an in vivo rituximab purge, autologous stem-cell transplantation (ASCT) and rituximab maintenance experienced superior progression free survival (PFS) compared to 40 matched, historical controls treated with conventional chemotherapy. We now report extended follow-up from this trial.

Methods: Eligible patients were treated with 4–6 cycles of CHOP (cyclophosphamide, doxorubicin, vincristine and prednisone) followed by high dose therapy (cyclophosphamide, carmustine and etoposide) and ASCT. Rituximab (375 mg/m2) was given once 5 days prior to stem cell collection (as an in vivo purge), and as two, 4-week maintenance courses 8 and 24 weeks after ASCT. Historical control patients matched for age, stage and gender were randomly selected from a lymphoma database maintained by the British Columbia Cancer Agency. Two control-patients were selected for every trial patient. All control patients were treated with either an anthracycline based-regimen, or a regimen containing fludarabine and cyclophosphamide.

Results: Median follow-up is 5.3 years for the experimental cohort and 10.1 years for the control cohort. In the ASCT cohort, 8 patients have relapsed and 4 patients have died, 2 of progressive disease, 1 of a presumed cardiac event 7 months post-ASCT, and one of hepatitis B reactivation 11 months post-ASCT. Five-year OS and 5-year PFS were superior in MCL patients treated with ASCT+ rituximab compared to matched, historical MCL patients treated with conventional chemotherapy (5y OS 80% vs. 38%, P=0.0017; 5y PFS 72% vs. 19%, P=0.0001). One patient developed myelodysplastic syndrome 5.5 years after ASCT, and one patient was diagnosed with breast cancer 4.8 years after ASCT.

Conclusions: With more than 5 years of follow-up MCL patients treated with ASCT plus rituximab continue to experience significantly improved OS and PFS compared to matched, historical controls.

Overall Survival Overall Survival
Prior Therapy with Rituximab (R) in Patients with Follicular Lymphoma (FL) Does Not Affect Relapse-Free (RFS) or Overall Survival (OS) after High Dose Therapy (HDT) and Autologous Stem Cell Transplantation (ASCT).
Authors: Kang Tyler Y. , Sweetenham John William , Thakkar Snehal G. , Rybicki Lisa A. , Brown Stacey , Dean Robert , Sekeres Mikkael A. , Advani Anjali , Sobecks Ronald , Kalaycio Matt , Pohlman Brad , Bolwell Brian J.
Published: October 2019

ASCT has been used in FL to achieve durable remissions either as consolidation therapy in first remission or as salvage therapy in the relapsed or refractory setting. The recent addition of R to chemotherapy regimens for FL has been shown to improve RFS and, in some studies, OS. The effectiveness of HDT and ASCT in pts with FL who have received prior therapy with R is unknown, although there is speculation that R may adversely affect outcome.

We retrospectively reviewed 128 consecutive patients with FL who underwent HDT and ASCT from 1/1994 through 12/2004. The analysis included 106 patients who were divided into No R and R groups. We excluded 19 patients who received R only as salvage therapy and three patients who received bone marrow rather than stem cells as their auto-graft. Patients were analyzed according to whether or not they had received an R-containing regimen prior to transplantation.

71 patients with No R therapy and 35 patients with R therapy were included. Characteristics between No R and R group are similar, with major difference being age, CD34+ dose, and length of follow-up. The No R group had a median OS that was not reached during follow up, with a median RFS of 49.9 months. The R group had a median OS of 63.2 months and a median RFS of 24.6 months. The OS and RFS were not significantly different between the groups. To control for potentially confounding factors between cohorts, we used propensity analysis to identify 22 patients among the R group who were well-matched to 22 patients in the No R group. Analysis was done for this well-matched cohort (Table 1), and OS and PFS were also found to have no significant difference (Figures 1 and 2).

In conclusion, we did not observe a difference in outcome in the effectiveness of HDT and ASCT between FL patients who received R therapy and those who did not. Prospective studies are required to confirm these results.

Table 1. Matched Groups by Rituximab Use No Rituximab Rituximab N % N % Age (years) mean +/− SD 49 +/− 8 59 +/− 7 median (range) 51 (28–69) 59 (49–75) Stage I/II 4 8.2 3 23.1 III 8 16.3 2 15.4 IV 37 75.5 8 61.5 # of Prior Chemotherap mean +/− SD 2.4 +/− 1.0 3.1 +/− 1.7 median (range) 2 (1–5) 2 (1–7) IPI Low, Low-Int 29 59.2 13 100 High-Int, High 20 40.8 0 0.0 Follicular Lymphoma - Matched Groups Follicular Lymphoma - Matched Groups Follicular Lymphoma - Matched Groups Follicular Lymphoma - Matched Groups
Influence of GST Gene Polimorphisms in the Develovement of Liver Sinusoidal Obstructive Syndrome in Patients with Multiple Myeloma Undergoing Hematopoietic Stem Cell Transplantation.
Authors: Martinez-Lopez Joaquin , Garcia-Efroom Guillermo , Martinez-Sanchez Pilar , Garcia-Sanz Ramon , Blade Juan , Sanchez-Godoy Pedro , Arriba Felipe , Garcia-Larana Jose Maria , Terol Maria Jose , Albiza Enriqueta , Montejano Laura , Grande Silvia , Rapado Inmaculada , Hernandez Belen , Miguel Jesus San , Lahuerta Juan Jose
Published: October 2019

Introduction: Liver metabolism has two fundamental types of enzymes. Phase two enzymes include GlutathioneS-transferases (GST), with three main variants: GST-M1, GST-T1 and GST-P1. Individual differences in these enzymes could influence their detoxificating capacity.

Aim: To study the influence of several genetic GST liver enzymes variants in the development of liver Sinusoidal Obstruction Syndrome (SOS) in patients with multiple myeloma (MM) that received BUMEL as conditioning regimen for Autologous Stem Cell Transplantation (ASCT).

Patients: 91 patients with MM - included in Spanish protocol Myeloma 2000 that had received BUMEL as conditioning schedule for ASCT - have been studied; 12 of them had developed SOS. 62 healthy individuals as well as 12 patients with monoclonal gammapathy of uncertain significance (MGUS) were also studied.

Methods: Three genotype variants of GST enzymes were studied: presence or absence of GST-M1, GST-T1 and GST-P1 polymorphism Ile105Val by real time PCR in light cycler v2 or ABI PRISM 7900HT thermocyclers. Associations between variables were studied by X2 and exact Fisher test. Logistic regression analysis was performed to calculate Odds ratios and relative risk of SOS development.

Results: Significant differences (p>0.0001) were found in the prevalence of homozygous genotype GST-P1 Ile105Val comparing the group of patients with MM and SOS (5 of 12, 41%) and the rest of MM patients without SOS (5 of 79, 6%). This genotype was present in 17% of healthy individuals and MGUS. The genotype absence of GST-M1 was observed in 33% (4/ 12) of MM patients with SOS, 46% (37/79) of MM patients without SOS (p=0.38), 56% of controls and 71% of MGUS. The genotype absence of GST-T1 was observed in 50% (6/ 12) of MM patients with SOS, 27% (22/79) without SOS (p=012), 19% (16/62) of healthy individuals and 21% of MGUS. 33% (4/12) of MM patients with SOS presented the absence of GST-T1 genotype and the presence of homozygous GST-P1 Ile105Val and none of the patients with MM without SOS (p=0,000) presented these genotypes. The only factor associated to development of SOS in regression study was the presence of genotype GST-P1 Ile105Val in homozygosis (OR 10.57 CI 2.45–45.6 p=0.002).

Conclusions: The genotype absence of GST-T1 together with the homozygous GST-P1 Ile105Val polymorphism, especially the latter, should be considered as new risk factors in the development of SOS in MM undergoing ASCT conditioning regimen with BUMEL.
Early Recovery of Thymus-Derived Naïve T Cells in Pediatric Patients (pts) Treated with Non-Myeloablative Allogeneic Peripheral Blood Stem Cell Transplantation (NMSCT) for Cancer.
Authors: Fry Terry J. , Rager Alison R. , Hakim Frances , Love Cynthia , Layton Paula , Fowler Daniel , Bishop Michael R. , Gress Ronald , Mackall Crystal L. , Wayne Alan S.
Published: October 2019

Background: Current SCT approaches consistently achieve rapid donor myeloid engraftment, but delayed immune recovery remains a significant obstacle and results in increased risk of infection and relapse. T cells are regenerated via 2 pathways, thymus-derived and peripheral expansion, processes for which IL-7 is critical. We postulated that non-myeloablative pre-transplant conditioning might preserve thymic function in pediatric SCT recipients thus enhancing thymus-derived naïve T cell regeneration.

Methods: We analyzed T cell subsets, T cell receptor excision circles (TREC), and IL-7 levels in peripheral blood after SCT in 21 pediatric pts with high-risk malignancies (median age 14, range 4–21). Fludarabine-based induction chemotherapy was administered for disease control and targeted CD4 count reduction. Pre-transplant conditioning consisted of cyclophosphamide (1,200 mg/m2/day) and fludarabine (30 mg/m2/day) × 4 days plus melphalan (100 mg/m2 × 1 dose in sarcoma pts). Grafts consisted of G-CSF mobilized unmodified peripheral blood stem cells from 5–6/6 HLA-matched first-degree relatives (median CD34 dose 11.7 × 10E6/kg, range 4.4–19.1; median CD3 dose 416 × 10E6/kg, range 228–815). Cyclosporine was used for GVHD prophylaxis.

Results: Donor-derived engraftment was rapid (absolute neutrophil count > 500/uL median day 9, range 8–11). Complete donor lymphoid chimerism (>95% by VNTR-PCR on CD3 sorted peripheral blood) was achieved in all by day 28. Immune recovery was brisk and sustained. Substantial numbers of naïve (CD45RA+/CD62L+) CD4+ and CD8+ T-cells were detected at day 28 (Fig 1). There was a steady increase in TREC from 3 to 12 months consistent with early, robust thymic-dependant T cell generation (Fig 2). This was not seen in adult pts treated on a parallel trial (data not shown). IL-7 levels were elevated and inversely correlated with T cell counts (r=−0.56, p<0.0001).

Conclusions: Targeted immune depletion and NMSCT results in rapid, sustained immune reconstitution in pediatric pts with malignancy. Preserved thymic function appears to contribute to naïve T cell recovery in this setting. We postulate that non-myeloablative conditioning is thymus sparing and that this, in combination with immune depletion-induced IL-7 elevation, promotes early thymic-derived lymphoid recovery. This approach may serve as a strategy to overcome the prolonged immunodeficiency commonly encountered after allogeneic SCT in pediatrics and might be used as a platform to direct allogeneic anti-tumor immune responses in high-risk childhood cancers.

Figure 1 Figure 1. Figure 2 Figure 2.
Outcomes of c Hematopoietic Stem Cell Transplantation (HCT) after Non-Myeloablative Conditioning in Relapsed, Refractory, or Transformed Indolent Non-Hodgkin Lymphoma (NHL).
Authors: Rezvani Andrew R. , Sandmaier Brenda M. , Storer Barry , Maris Michael , Agura Edward , Maziarz Richard , Wade James C. , Chauncey Thomas , Forman Stephen J. , Niederwieser Dietger W. , Shizuru Judith , Langston Amelia , Pulsipher Michael , Storb Rainer F. , Maloney David G.
Published: October 2019

Sixty-two patients (pts) with chemotherapy-refractory indolent or transformed NHL were treated at 10 centers with allogeneic HCT from related (n=34) and unrelated (n=28) donors after 2 Gy total body irradiation with or without fludarabine. Diagnoses included follicular lymphoma (FL) (n=54, including 10 with grade 3 FL), small lymphocytic lymphoma (n=6), and marginal zone lymphoma (n=2). Median age was 54 years (range 33–66 years), and median time from diagnosis to HCT was 4.4 years (range 0.5–18.5 years). Sixteen pts had histologically documented transformation to diffuse aggressive lymphoma prior to HCT. Twenty-seven pts (44%) had failed autologous HCT. Disease status at the time of HCT was complete response (CR, n=16), partial response (PR, n=22), refractory (n=13), untested relapse (n=9), or unknown (n=2). Eleven of the 28 unrelated donor/recipient pairs (39%) had HLA mismatches: 2 at a single allele, 7 at a single antigen, and 2 at an antigen and an allele. One pt had non-fatal graft rejection from a 1-antigen-mismatched unrelated donor. Median follow-up of survivors after HCT was 36.6 months (range 2.3–60 months). Responses (CR [n=18] and PR [n=7]) were seen in 25 of 44 (57%) pts with evaluable disease prior to HCT, while 5 had stable disease, 9 progressed, and 5 were not evaluable due to early non-relapse mortality (NRM) on d27–d108. Two of 16 pts (13%) transplanted in CR relapsed; one was treated with donor lymphocyte infusion and achieved a persistent CR. The incidences of acute GVHD grades II–IV, III–IV, and chronic GVHD were 63%, 19%, and 53%, respectively. At 3 years, the risks of relapse/progression and NRM were 19% and 42%, respectively. There was a trend toward increased mortality with unrelated donors (HR 1.87 [0.9–3.7, p=0.08]). Progression-free and overall survival (PFS and OS) were significantly better in the non-transformed group (see tables 1 and 2).

Table 1. Outcomes Non-transformed Transformed Relapse 6/46 (13%) 6/16 (38%) NRM 20/46 (43%) 6/16 (38%) 3-year OS 24/46 (52%) 4/16 (25%) 3-year PFS 20/46 (43%) 4/16 (25%) Table 2. Hazard Ratios (HR) for Transformed vs. Non-Transformed Pts HR (95% CI) p All-cause Mortality 2.39 (1.2–4.9) 0.02 Relapse/progression 4.75 (1.5–15) 0.01 Grade 3+ acute GVHD 1.84 (0.5–6.3) 0.35 Extensive chronic GVHD 1.96 (0.8–5.0) 0.18 Figure Figure

Allogeneic HCT after non-myeloablative conditioning can produce durable responses and prolonged survival in pts with refractory indolent or transformed NHL. Pts transplanted before histologic transformation had significantly better outcomes. Future efforts will focus on reducing NRM and identifying optimal timing of HCT.
Serum Amyloid A (SAA) as a Molecular Marker for Pre-Engraftment Immune Reactions (PIR) after Cord Blood Transplantation.
Authors: Morita-Hoshi Yuriko , Heike Yuji , Ohsaki Yuka , Shiwa Mieko , Masuoka Kazuhiro , Wake Atsushi , Taniguchi Shuichi , Tobinai Kensei , Takaue Yoichi
Published: October 2019

〈Background and Methods〉: High-grade fever prior to engraftment without any apparent signs of infection, mimicking hyperacute GVHD or engraftment syndrome, is often observed in patients who undergo cord blood transplantation (CBT). This “pre-engraftment immune reaction (PIR)” peaks at around day 9 of CBT, and is often accompanied by high-grade fever, skin rash and weight gain. Although PIR responds well to corticosteroid therapy, the prolonged use of steroid often causes an increased incidence of infectious complications, leading to significant treatment-related mortality, particularly in the elderly. It has been speculated that cytokines induced by the initial immune/inflammation reaction are the primary cause of PIR, but no confirming data are available. To clarify this point, we evaluated the protein expression profile of serum in CBT recipients using a surface-enhanced laser desorption/ionization time-of-flight mass spectroscopy (SELDI-TOF MS) system and found marker candidates in PIR after CBT.

〈Results〉: Thirty-eight blood samples were taken from 13 CBT recipients at 3 different occasions, i.e.

afebrile period prior to PIR onset, onset of fever and resolution of fever, to compare the relative protein expression levels.

Samples were analyzed in 56 spectra by changing the elution conditions with 4 kinds of chips (IMAC30, CM10, H50, Q10), 5 different rinse conditions and 3 kinds of energy absorption molecules. Six candidate protein peaks that commonly increase at the time of PIR with molecular masses of 8611Da, 8642Da, 11452Da, 11512Da, 11539Da and 11669Da were identified. The 11kDa protein peaks that bind to a Q10-anion exchange chip were selected for further analysis. Two 11kDa protein peaks were eluted at pH9 and pH4, but since the proteins eluted at pH4 included albumin which overlapped the candidate protein peak, the elution fraction at pH9 was used for purification and identification. Proteins eluted at pH9 were separated by SDS-PAGE. After in-gel digestion of the 11kDa band by trypsin, the digested peaks were analyzed by PCI Qstar MSMS and the protein was determined using the ProFound database. The results revealed that the 11kDa protein was an N-terminal portion of serum amyloid A (SAA). The SAA level was measured by ELISA in the same sample that was assessed by SELDI-TOF MS. The mean SAA level prior to fever onset was 14 (3–51) μg/mL, and this increased to 883 (40–2470) μg/mL at the time of PIR and decreased to 45 (8–126) μg/mL after resolution of the fever.

〈Conclusion〉: A specific marker for PIR after CBT was identified by an SELDI-TOF MS system. SAA increased by 10- to 100-fold at the time of PIR, and thus may become a useful tool for the diagnosis of PIR.

Figure Figure
Unrelated Donor Hematopoietic Cell Transplantation after Non-Myeloablative Conditioning for Patients with High Risk Multiple Myeloma.
Authors: Sorasio Roberto , Giaccone Luisa , Patriarca Francesca , Montefusco Vittorio , Guidi Stefano , Busca Alessandro , Scime’ Rosanna , Console Giuseppe , Milone Giuseppe , Marotta Giuseppe , Dominietto Alida , Rotta Marcello , Falda Michele , Bacigalupo Andrea , Bosi Alberto , Corradini Paolo , Fanin Renato , Pollichieni Simona , Bruno Benedetto , Boccadoro Mario
Published: October 2019

Allografting can induce long-term molecular remissions and possibly cure in myeloma patients. The recent development of non-myeloablative conditionings has reduced the transplant-related mortality (TRM) typically associated with myeloablation and extended the eligible age for transplantation. Moreover, high response rates are reported especially when allografting is preceded by cytoreductive high dose chemotherapy. We investigated the feasibility of unrelated donor non-myeloablative transplantation as either part of the initial treatment plan or as salvage treatment in heavily pre-treated patients. Twenty-two patients underwent non-myeloablative allografting, 10 as part of their initial treatment and 12 at disease relapse. Donors were matched for HLA-A, B, C, DRB1 and DQB1 by high-resolution typing. Only one single class I allele disparity was allowed. Conditioning regimen consisted of fludarabine 90 mg/m2 and 2 Gy total body irradiation. GVHD prophylaxis included oral cyclosporine (CyA) and mycophenolate mofetil (MMF). CyA was administered at 6.25 mg/Kg every 12 hours from day -3; levels were targeted to the upper therapeutic range (450–500 ng/ml, Abbott TDX, Abbott Park, IL) for the first month post-transplant. In the absence of GVHD, CyA was tapered from day +100 and discontinued on day +177. MMF was administered from day 0 after PBSC infusion to day +40 at 15 mg/Kg every 8 hours, and then tapered till day +96. Twenty/22 (91%) patients readily engrafted. Two patients experienced graft failure and eventually recovered autologous hematopoiesis. After a median follow up of 11 months (3–27), TRM was 18% and 16/22 patients (73%) are alive. Deaths occurred in 10% of patients transplanted upfront and in 42% of those transplanted at relapse: 3 patients died from infections, 1 from hemolytic uremic/ thrombotic thrombocytopenic purpura syndrome, and 2 from disease progression (both were transplanted at relapse). Ten/20 engrafted patients (50%) had grade II–IV acute GVHD (10% grade III–IV), and 59% had extensive chronic GVHD. Overall response rate was 60% (including 20% CR): 78% in patients transplanted upfront (no disease progression observed) and 45% in those transplanted at relapse. In the two groups, progression-free and one year event-free survival were 100% and 44% (p<0.025), and 90% and 28% respectively (p<0.005).

Unrelated donor non-myeloablative allografting is feasible with relatively low TRM and high response rate. Graft-versus-myeloma effect appears to be more efficient when patients are treated soon after diagnosis. Longer follow-up is needed to assess response duration.
Factors Influencing Short Term In Vivo Biodistribution of Human Bone Marrow Derived Mesenchymal Stem Cells (hMSCs) in a Xenogeneic Animal Model.
Authors: Kyriakou Charalampia , Rabin Neil , Nathwani Amit , Pizzey Arnold , Linch David C. , Yong Kwee
Published: October 2019

Human MSCs are potential agents for tissue regeneration, enhancing haemopoietic stem cell transplantation and delivering genes of therapeutic interest. To successfully implement any of these therapeutic strategies, we need a better understanding of the tissue localisation of systemically administered hMSCs and of the factors that critically influence their biodistribution. Bone marrow (BM) derived hMSCs were isolated from normal donors, cultured using previously described protocols and characterised by phenotype and multi-lineage differentiation. HMSCs were labelled with PKH-26 and injected by tail vein into b2m/NOD/SCID animals (2x106 cells /animal). Analysis of murine tissue mononuclear cells by flow cytometry 24 hours later consistently detected a proportion of transplanted cells in the BM (327 ± 31), spleen (645 ± 147) and lungs (1485 ± 138) in all experiments. This was also confirmed by flow cytometry using human-specific HLA-Class I mAb and FISH analysis. Homing of infused hMSCs to the BM and spleen of b2m/NOD/SCID animals was significantly higher in younger (<10 weeks) when compared with older (>10 weeks) animals (p<0.0001). [BM (424 ± 24 vs 209 ± 20), spleen (1175 ± 154 vs 292 ± 30), lungs (2760 ± 321 vs 1225 ± 269) respectively]. Prolonged ex vivo expansion of hMSCs also impaired their homing. Homing to the BM and spleen was significantly lower (p<0.022, and p<0.015 respectively) for late passage (> P14) when compared with earlier passage hMSCs (< P10). Lentiviral transduction of hMSCs with eGFP did not alter homing when compared to unmanipulated cells.

Tissue homing was markedly enhanced by prior irradiation of animals (325Gy). Numbers of PKH positive cells detected by flow cytometry were significantly higher in the BM of irradiated recipients (425.3 ± 42 vs 110 ± 20 for unirradiated mice, p=0.001). Irradiation also increased homing to the lungs (4722 ± 1220 vs 495 ± 70, p=0.0327). Overexpression of CXCR4 by lentiviral gene transfer in hMSCs significantly enhanced their migration to SDF-1 in vitro. CXCR4 overexpression did not influence short term homing to the BM and other tissues of unconditioned b2 m NOD/SCID. In contrast, CXCR4 transduced hMSCs demonstrated significantly higher levels of homing (compared with eGFP-control transduced hMSCs) when transplanted into irradiated b2 m NOD/SCID. This was particularly marked in the lungs (p=0.007).

Conclusion: In vivo homing of hMSCs is critically influenced by animal age, length of ex vivo cell manipulation and is enhanced in the irradiated-stressed animal model. Overexpression of CXCR4 does not significantly alter hMSCs homing in the unconditioned animal model but does so in the irradiated recipients. Identifying these factors and strategies to improve tissue homing is of great importance for the successful application of hMSCs in cell and gene therapy.
Fibrinogen Enhances Homing-Related Responses of CD34+ by Incorporating Membrane Type1-Matrix Metalloproteinase into Membrane Lipid Rafts.
Authors: Shirvaikar Neeta , Turner A. Robert , Ratajczak Mariusz Z. , Janowska-Wieczorek Anna
Published: October 2019

Clinical studies have shown that transplanted mobilized peripheral blood (mPB) hematopoietic stem/progenitor cells (HSPC) home faster to the bone marrow (BM) than harvested BM or cord blood cells. We previously showed that fibrinogen has a priming effect on HSPC homing by enhancing the chemotactic responses of CD34+ cells towards a low SDF-1 gradient by incorporating CXCR4 into membrane lipid rafts (Blood2005; 105:40). In this study we further investigated the mechanism of priming by determining the effect of fibrinogen on matrix metalloproteinases (MMPs) expressed by CD34+ cells, particularly MT1-MMP which is localized on the leading edge of migrating cells and not only activates latent MMPs but by itself degrades several extracellular matrix (ECM) components such as collagens, laminin, fibrin and fibronectin.

We found that fibrinogen

highly upregulated MT1-MMP protein expression (Western blot) in CD34+ cells as well as proMMP-2 and proMMP-9 secretion (zymography); primed trans-Matrigel chemoinvasion of CD34+ cells towards a low SDF-1 gradient (20 ng/mL), which was inhibited by epigallocatechin-3-gallate, a potent inhibitor of MT1-MMP; and stimulated MMP-2 activation in co-cultures of stromal cells (BM fibroblasts and HUVEC) with CD34+ cells.

Moreover, we demonstrate by confocal microscopy, for the first time, that in CD34+ cells MT1-MMP is localized in the GM1-fraction of lipid rafts where it co-localizes with CXCR4; this co-localization is enhanced when CD34+ cells are stimulated with fibrinogen. Furthermore, disruption of lipid raft formation by the cholesterol-depleting agent methyl-b-cyclodextrin inhibited MT1-MMP incorporation into membrane lipid rafts and also trans-Matrigel chemoinvasion of CD34+ cells towards an SDF-1 gradient. Thus we conclude that fibrinogen enhances homing-related responses of CD34+ cells towards SDF-1 by increased incorporation and co-localization of CXCR4 and MT1-MMP in membrane lipid rafts. Further, we postulate that while the presence of CXCR4 in lipid rafts allows the cells to better sense the SDF-1 chemokine gradient, the upregulated MT1-MMP in the lipid rafts facilitates their migration through the ECM and possibly towards the BM niches.
Prevention and Reversal of Lupus in NZB/NZW Mice by Transplantation of Purified Allogeneic Hematopoietic Stem Cells (HSC) with Non-Myeloablative Conditioning (NMT).
Authors: Smith-Berdan Stephanie , Gille Daphne , Christensen Julie L.
Published: October 2019

Systemic lupus erythematosus (SLE) is a multifactorial autoimmune disease. Patients’ refractory to therapy may be considered for hematopoietic stem cell transplantation. Using lupus prone female NZB x NZW (NZBW) mice, we tested the ability of highly enriched, allogeneic HSC to prevent and reverse autoimmune symptoms with FACS purified haplo-identical allogeneic HSC. Ablative conditioning: 92 animals were given lethal TBI (14.5 Gy) and divided among 4 groups (Table 1). Urine and serology tested monthly, final time point before death tabulated. Transplantation with either syngeneic HSC or WBM accelerated disease in these mice, resulting in a rate of death exceeding age matched controls. Allogeneic transplanted mice had significantly greater survival above all groups (p= 0.0243). Proteinuria, elevated levels of circulating immune complexes (CIC), and auto-antibodies to dsDNA, nuclear antigens (ANA) and histones were lower in allo-HSC animals compared to the other ablative conditioning groups (p≤ 0.0001). Overall survival (OS) in allo-HSC animals was still unexceptional, possibly due to regimen related toxicity (TRM).

Table 1: Ablative age@TX=75 days Age Matched Control Syngeneic WBM Syngeneic HSC Allogeneic HSC N 15 21 28 28 OS@420 days of age 20% 0% 0% 53% Proteinuria 100% 74% 75% 15% CIC 93% 81% 100% 25% Anti-dsDNA 100% 91% 100% 39% Anti-ANA 100% 86% 93% 39% Anti-Histone 93% 91% 100% 46%

NMT conditioning: To determine if we could attenuate disease in NZBW mice already progressing into lupus-like disease with transplantation of allogeneic, purified HSC and reduce TRM, we developed a non-myeloablative conditioning protocol (2x5 Gy TBI + ATG + a-ASIALO-GM1) achieving an average mixed chimerism of 50%. Animals were treated at ~241 days with established symptoms of lupus (Table 2). While the group receiving conditioning alone, had a slight survival advantage over age matched control mice, the transplanted mice had greatly increased OS with 70% living well beyond 500 days of age (>250 days from transplant). Allo-HSC mice showed reversal or stabilization of their lupus symptoms including proteinuria, CIC, dsDNA and histone.

Table 2: NMT age@Tx=241 days Age Matched Control Allogeneic HSC Conditioned Only N 10 33 30 OS@500 days of age 0% 70% 0% Proteinuria@Tx 20% 49% 47% Final Proteinuria 100% 39% 67% CIC 67% 50% 74% Anti-dsDNA 85% 15% 44% Anti-Histone 100% 37% 66%

Conclusions:

Ablative and NMT transplant can treat lupus; OS after NMT exceeds ablative conditioning; Induction of mixed chimerism with purified allogeneic HSC using NMT conditioning treats established lupus.

The ability of pure HSC transplant and establishment of durable mixed chimerism to reverse established lupus makes it a reasonable strategy to test in man.
Repertoire Analysis of Ex Vivo Polyclonally Expanded Regulatory T Cells.
Authors: Zilberberg Jenny , Goldgirsh Kira , Korngold Robert , Friedman Thea M.
Published: October 2019

CD4+CD25+ regulatory T cells (Treg) are essential for the maintenance of self-tolerance and have also been implicated in the control of alloreactive immune responses. Several studies using murine models of graft-vs.-host disease (GVHD) have shown that addition of equivalent numbers of Treg to the donor T cell inoculum at time of hematopoietic stem cell transplantation can significantly reduce the incidence of GVHD. In addition, in an MHC-matched, minor histocompatibility disparate model, the infusion of Treg ten days post-transplantation was shown to ameliorate the progression of GVHD while permitting a graft-versus-leukemia effect. However, because Treg constitute <5% of peripheral CD4+ T cells in humans, the use of freshly isolated Treg to treat and/or prevent GVHD, as well as other diseases in the clinical situation, is limited. Therefore, much effort is now under way to expand Treg in order to have sufficient numbers for therapeutic use. There is little available information regarding the repertoire complexity of ex vivo, polyclonally expanded regulatory T cells. We hypothesize that like their CD4+CD25− T cell counterparts, the diversity of the Treg T cell receptor (TCR) repertoire will also be complex. To this end, CD4+CD25− and CD4+CD25+ T cells from B10.BR mice were purified using fluorescence activated cell sorting; both populations were polyclonally expanded using CD3/CD28 paramagnetic microbeads in combination with high levels (100 IU/ml) of hrIL-2. After achieving a greater than 50 fold expansion, RNA from 1–1.5×107 cells was isolated for RT-PCR. The complexity of the T cell repertoire of expanded CD4+CD25− and CD4+CD25+ was determined using TCR Vb CDR3-size spectratype analysis. The PCR products were run on a sequencing gel and analyzed by the GeneMapper Software from Applied Biosystems. This comparison revealed that the number of resolvable Vb families is more heterogeneous in the CD25− populations. Whether this reflected a lack of complexity in the regulatory repertoire warrants further investigation. However, for the resolvable Vb families there were no significant differences in the complexity indexes between these two groups.
Imaging the Differential Engraftment of GFP-Expressing Spleen and Bone Marrow Cells in Various Organs after Reduced-Intensity Stem Cell Transplantation in Mice Model.
Authors: Heike Yuji , Yang Zhijian , Ma Huaiyu , Hoffmann Robert , Morita Yuriko , Soeda Atsuko , Makiyama Hiroaki , Tobinai Kensei , Takaue Yoichi
Published: October 2019

[Background] Reduced-intensity allogeneic stem cell transplantation is of critical importance in the treatment of hematological malignancies. The object of this experiment is to establish fludrabine-based reduced-intensity allogeneic stem cell transplantation model using donor cells labeled with GFP and image the engraftment process of donor cells in various organs.

[Materials and Methods] GFP transgenic C57/BL6 mice (GFP-Tgm) were used as donors and C57/BL6 × DBA F1 mice (BDF1) were used as recipients. Recipient mice were pretreated with fludarabine (Flu) 150 mg/kg/day × 6 days i.p. and cyclophosphamide (CPA) 150 mg/kg/day × 2 days i.p. On day 0, 107 GFP splenocytes (Group 1) and GFP bone marrow cells (Group 2) were injected in the tail vein. Whole body and intravital imaging were used to visualize the migration of GFP-Tgm cells into various organs including the brain, femur, intestine, liver, lung, ovary, pelvic bone, ribs, skin, skull, spine, spleen and uterus on days-7 and -14. The macro images were obtained with the Olympus OV100 Small Animal Imaging System. GFP-Tgm cell migration, particularly CD34+ cells in the various organs was also analyzed by flow cytometry (FACS) using APC-labeled anti-CD34 monoclonal antibodies.

[Results and Discussion] On day-7, the migration of donor GFP-Tgm cells in peripheral lymph nodes, intestine, lung, ovary, skin and uterus were detected in both groups. Migration of GFP-Tgm cells in the femur, pelvic bone, ribs and skull were clearly detected in Group 2, but not in Group 1. On day-14 GFP donor cells were imaged in the lung, ovary, skin and uterus both groups. However, GFP-Tgm cells were no longer imaged in the intestine in Group 2 except in Payer patches. In both groups the GFP-Tgm cells were strongly detected in the femur, pelvic bone, skull and spine on day-14. We also analyzed the migration of CD34+ GFP-Tgm cells by FACS analysis. On day-14, the percentage of GFP-Tgm cells in the bone marrow and spleen was, respectively, 14% and 53% in Group 1, and 10% and 13% in Group 2. The percentage of CD34+ GFP-Tgm cells among total CD34+ cells in the bone marrow was 10% in Group 1 and 14% in Group 2, and in the spleen was 24% and 19%, respectively. Those results suggested that in this model, donor bone marrow and spleen cells, but not purified CD34+ cells, have different engraftment kinetics in various organs including the intestine, which is a target organ for graft-versus host disease. These results clearly suggest that caution should be paid to evaluate engraftment kinetics of infused cells, at least in mice model.
Adoptive Infusion of Alloreactive Donor NK Cells Reduces GVHD, Mediates Anti-Tumor Effects and Prolongs Survival in Recipients of MHC-Matched Hematopoietic Cell Transplantation.
Authors: Lundqvist Andreas , McCoy Philip , Samsel Leigh , Smith Aleah , Srivastava Shivani , Harashima Nanae , Yokoyama Hisayuki , Berg Maria , Childs Richard
Published: October 2019

Alloreactive natural killer (NK) cells with killer immunoglobulin-like receptor (KIR) ligand incompatibility have been implicated to reduce graft-vs-host disease (GVHD) and leukemia relapse in humans undergoing MHC-mismatched T-cell depleted allogeneic hematopoietic cell transplantation (HCT). Analogous to human NK KIR, murine NK cells express subgroups of MHC-specific receptors of the C-type lectin superfamily, termed Ly49, that regulate their function. In MHC-mismatched HCT, the infusion of alloreactive Ly49-mismatched NK cells decreases the incidence of GVHD. We investigated if an infusion of alloreactive NK cells would reduce GVHD and further mediate anti-tumor effects in mice undergoing MHC-matched allogeneic HCT. Balb/c mice were injected intravenously with 105 RENCA tumor cells. Ten days following tumor injection, mice were irradiated (950cGy) and transplanted with 15×106 and 8×106 splenocytes and bone-marrow cells respectively from MHC-matched B10.d2 donors. Five days following transplantation, recipients were given a single infusion of either 0.5–1×106 Ly49 ligand-matched (Ly49G2; non-alloreactive to Balb/c) or Ly49 ligand-mismatched (Ly49C; alloreactive to Balb/c) donor NK cells. Recipients of Ly49 ligand-mismatched NK cells had a significant reduction in the incidence of GVHD (p<0.01-figure), and prolonged survival (median 84 vs 39 days; p<0.01-figure) compared to HCT recipients not receiving NK cells. Recipients of Ly49 ligand-matched NK cells had the same incidence of GVHD and similar survival compared to controls not receiving NK cells. Pulmonary tumor burden was significantly (p<0.01) lower in recipients of Ly49-mismatched or Ly49-matched NK cells compared to mice not receiving NK cells. These data provide in vivo evidence that a single infusion of alloreactive donor NK cells can prolong survival by both reducing GVHD and mediating anti-tumor effects following MHC-matched allogeneic HCT.

Figure: Significant reduction in GVHD and improved survival in RENCA tumor bearing mice undergoing an allogeneic HCT that received an infusion of alloreactive NK cells (Ly49C:thick line) compared to transplant recipients receiving non-alloreactive NK cells (Ly49G2:thin line) or no NK cells (dotted line) Figure:. Significant reduction in GVHD and improved survival in RENCA tumor bearing mice undergoing an allogeneic HCT that received an infusion of alloreactive NK cells (Ly49C:thick line) compared to transplant recipients receiving non-alloreactive NK cells (Ly49G2:thin line) or no NK cells (dotted line)
Allogeneic Stem Cell Transplantation for MDS and sAML Following Reduced Intensity Conditioning and Preemptive Donor Lymphocyte Transfusion.
Authors: Kolb Hans-Jochem , Schmid Christoph , Tischer Johanna , Haussmann Andreas , Rank Andreas , Schleuning Michael , Ledderose Georg
Published: October 2019

Myelodysplastic syndrome (MDS) and secondary acute myeloid leukemia (sAML) are diseases of the hematopoietic stem cell that are potentially curable by allogeneic stem cells transplantation (ASCT). MDS and sAML are most frequent in elderly patients with significant co-morbidity. Here we report our results with reduced intensity conditioning regimen and preemptive donor lymphocyte transfusions that have been successful in patients with advanced and refractory AML. Sixty seven patients were treated for MDS and 90 patients for sAML. The FLAMSA regimen consisted of a 4 day course of chemotherapy (Ara-C 2g/m2, Fludarabin 30 mg/m2, and amsacrin 100 mg/m2) followed by 3 days rest and reduced intensity conditioning with 4 Gy total body irradiation (TBI), cyclophosphamide (CY) +/− antithymocyte globulin (ATG). Overall survival at 10 years was 50% for MDS and 26% for sAML (p=0.014, log rank). In the MDS group most patients had preleukemic forms (IPSS 3 and 4). Survival at 5 years was 58% with FLAMSA, 44% with myeloablative conditioning with TBI and 67% with Busulfan (BUS). Non-relapse mortality at 5 years was 41% with FLAMSA, 20% for BUS and 50% for TBI. The relapse or progression rate was 0% for FLAMSA, 24 % for BUS and TBI respectively. In sAML the 5 year survival was 17% for FLAMSA, 57 % for BUS and 30% for TBI. Non-relapse mortality was 60% for FLAMSA, 36% for BUS and 66% for TBI, whereas the relapse rate was 62% for FLAMSA, 12.5 % for BUS and 37% for TBI (p=0.04). In multivariate analysis adjusted for disease, conditioning treatment and source of stem cells – marrow vs. mobilized blood – differences were not significant and the only significant prognostic factor was age less than 30 years (p=0.003). Therefore patients of the age of 60 years and older with AML were treated with FLAMSA containing BUS (8mg/kg in two days) instead of 4 Gy TBI. The overall survival at 2 years was 64%, 61% with FLAMSA-TBI and 70% with FLAMSA-BUS. The non-relapse mortality was 9% for FLAMSA-BUS and 37% for FLAMSA-TBI; the relapse rate was 25% at 2 years. At day 120 patients received donor lymphocytes (DLT) in 3 escalating doses from 1 × 106/kg, 5 × 106/kg on day 150 and 1 × 107/kg on day 180, if they had no GVHD, infection or relapse. Preemptive DLT appear to prevent relapse when compared to a historical control and contemporary patients not given DLT. We conclude from these data that MDS/sAML may have a different biology in young patients, and that in the elderly we can optimize the treatment with FLAMSA-BUS as a dose reduced regimen and preemptive immunotherapy for maintenance of remission.
SNP12 and SNP13 Variants of NOD2/CARD15 Gene of Donor and Recipient as Independent Risk Factors for Severe Intestinal and Pulmonary GVHD after Allogeneic Hematopoietic Stem Cell Transplantation.
Authors: Holowiecka-Goral Alexandra , Wojnar Jerzy , Giebel Sebastian , Grudziecka Elzbieta , Oczko Malgorzata , Jarzab Barbara , Sankowska Monika
Published: October 2019

Background: Presence of any of single nucleotide polymorphism (SNPs) of the NOD2/CARD15 gene have been recently proven to correlate with higher rate of GvHD after allogeneic hematopoietic stem cell transplantation - alloHSCT (Holler E et al; Blood2006,107,4189). Mutated variants lead to impaired antibacterial response. As NOD2/CARD15 is expressed not only in monocytets/macrophages but also in both intestinal and bronchial epithelial cells these organs seem to be a major target for developing advanced GvHD.

Patients and methods: The impact of each single donor (D) and recipient (R) SNPs (8,12,13) of NOD2/CARD15 gene on the incidence of acute GvHD (grade II-IV, grade III-IV, intestinal), chronic GvHD (overall, extensive, pulmonary), survival, and non-relapse mortality was evaluated in a setting of 72 patients treated with alloHSCT in single BMT center in Katowice between Jan 2000 and Jun 2005. Median patient age was 33 (11–52) years. In 70/72 cases hematological malignancies were indication for alloHSCT. Conditioning regimen was myeloablative in all cases, bone marrow was used as the only source of stem cells in 74% of patients. GVHD prophylaxis consisted of CsA, Mtx and, in case of URD-HSCT - antithymocyte globulin.

Results: In univariate and multivariate analysis, including other potential risk factors, the presence of SNP12 in the recipient was associated with increased incidence of intestinal acute GVHD (75% vs. 29%; RR 4,37, p=0,03), overall chronic GVHD (100% vs. 50%, RR 4,72; p=0,003), extensive chronic GVHD (80% vs. 24%; RR 5,12; p=0,02), and pulmonary chronic GVHD (47% vs. 9%; RR 5,97; p=0,02). SNP13 in the recipient resulted in increased incidence of grade III–IV acute GVHD (45% vs. 12%; RR 4,66; p=0,01), intestinal acute GVHD (64% vs. 24%; RR 4,21, p=0,005), and extensive chronic GVHD (55% vs. 25%; RR 3,59; p=0,03). SNP13D in the donor contributed to increased risk of pulmonary chronic GVHD (36% vs. 8%; RR 6,58; p=0,01). In this single centre analysis we were not able to demonstrate the impact of NOD2/CARD15 SNPs on survival.

Conclusion: The presence of particular mutations of NOD2/CARD15 in the recipient and/or the donor is associated with increased risk of severe acute and chronic GVHD. In particular, intestines and lungs appear to be target organs of these complications. Our findings may contribute to optimization of immunosuppressive and gastrointestinal decontamination regimens based on individual risk assessment for GVHD.