bluuebunny/crossref_metadata_2025 huggingface dataset:
Computed Tomography (CT) Predicts Response Duration in Patients with Chronic Lymphocytic Leukemia (CLL) in Complete Response (CR) by Conventional, NCI-Working Group, Criteria.
Authors: Bosch Francesc , Muntañola Ana , Ferrer Ana , González Marcos , Gine Eva , Abella Eugenia , Gardella Santi , Briones Javier , González-Barca Eva , Escoda Lourdes , Pérez-Ceballos Elena , Asensi Antoni , Sayas Maria José , Besalduch Joan , Montserrat Emili
Published: October 2019

Imaging studies are not routinely performed to evaluate response to therapy in patients with CLL. To ascertain whether CT scans should be part of the maneuvers to assess response in patients with CLL we have investigated response duration according to CT results in 34 previously untreated patients (median age, 56 years; range, 38 to 65 years; males: 79%) uniformly treated with FCM (fludarabine 25 mg/m2 i.v. on days 1 to 3, cyclophosphamide 200 mg/ m2 on days 1 to 3, and mitoxantrone 6 mg/m2 i.v. on day 1, given at a 4-week intervals up to six courses) as part of a phase II study. Out of 69 patients included in the study, 64% achieved CR as defined by the NCI-Working Group criteria. Of those, 30% had abdominal lymphadenopathy as assessed by CT scans. The median follow-up of the series was 30 months. As shown in the figure, response duration was significantly shorter (54% at 20 months) in CR patients with abnormal CT (CT+ CR) than in those with no detectable lymphadenopathy (CT- CR) (95% at 20 months). Interestingly, response duration in patients with CT+ CR was not different from that of patients in PR (54% vs. 42% at 20 months, respectively; p=NS). These results demonstrate that residual abdominal lymphadenopathy negatively influences response duration in patients with CLL in whom by all other criteria a CR has been obtained. Therefore, CT scans should be performed in patients having achieved CR by conventional, NCI-Working Group criteria, and patients with residual disease by CT should be considered in PR rather than in CR.

Figure Figure
Dasatinib (SPRYCEL®) in Patients (pts) with Philadelphia Chromosome-Positive Acute Lymphoblastic Leukemia Who Are Imatinib-Resistant (im-r) or -Intolerant (im-i): Updated Results from the CA180-015 ‘START-L’ Study.
Authors: Dombret Herve , Ottmann O.G. , Rosti G. , Simonsson B. , Larson R.A. , Gollerkeri A. , Apanovitch A. , Radich J. , Coutre Steven
Published: October 2019

Dasatinib (SPRYCEL®, formerly BMS-354825) is a novel, oral, multi-targeted kinase inhibitor of BCR-ABL and SRC. Relapsing pts with Philadelphia chromosome-positive acute lymphoblastic leukemia (Ph(+) ALL) who have been previously treated with chemotherapy including im have a very poor prognosis. START-L is an open label, multi-center, global phase-II study, which treated 46 im-r or im-i pts with Ph(+) ALL from January through June 2005. Dasatinib was given orally, 70 mg twice daily (BID) with escalation to 100 mg BID for poor response or reductions to 50 mg and 40 mg BID for toxicity. Response was assessed on weekly blood counts and monthly bone marrow exams, including cytogenetics. Mutation analysis was conducted at baseline and at end of study. Quantitative PCR was carried out pretreatment and at the time of complete cytogenetic response (CCyR). At the time of this update, all 46 pts had a minimum of 9 months (mo) of follow-up. Of these, 44 (96%) were im-r; due to the small number of im-i pts, data for all pts are presented. 59% of patients were male and median age was 48 years. The median time from initial Ph(+) ALL diagnosis to first dose was 18 mo. All patients had been pretreated with im including 46% with 600 mg per day or more, and 52% whose duration of im treatment was more than 12 mo. 37% of the pts had received a stem cell transplant. At baseline, 67% of pts had WBC <2000/mm3, 74% had platelets <100 × 103/mm3, and 24% had extramedullary disease outside of the spleen. In the 40 pts with baseline mutation data, im-resistant BCR-ABL mutations were observed in 78%, one with T315I. The median duration of therapy was 3.0 mo (range 0.03–16.5) for all pts and was 13.0 mo (9.2–16.5) for pts still on study. The overall complete hematologic response rate was 35%. The major cytogenetic response was 57%, including 54% CCyR. The major hematologic response (MHR) in the 31 pts with baseline mutations was 45%. The median duration of MHR was 11 mo and the median progression-free survival was 3.7 mo (95% CI 1.6–6.1). Grades 3 and 4 thrombocytopenia occurred in 13% and 67%, respectively and grades 3 and 4 neutropenia occurred in 27% and 52% of pts, respectively. Most frequent non-hematologic toxicities included diarrhea in 30% (grade 3–4, 7%), nausea in 22% (no grade 3–4), pyrexia in 22% (grade 3–4, 2%), and pleural effusion in 22% (grade 3–4, 7%) of pts. Dose was reduced in 30% and interrupted in 43% of pts, primarily due to non-hematologic toxicities. Dasatinib has important efficacy in this Ph(+) ALL pt population. Updated efficacy (including molecular response), safety, and mutational analysis data will be presented at the meeting.
Allo-Reactive NK Cells after HLA-Matched Allogeneic Hematopoietic Stem Cell Transplantation.
Authors: Hasenkamp Justin , Borgerding Andrea , Chapuy Bjoern , Wulf Gerald , Missal Inga , Jung Wolfram , Truemper Lorenz , Glass Bertram
Published: October 2019

Allo-reactive natural killer (NK) cells frequently occur early after haplo-mismatch hematopoietic stem cell transplantation (HSCT) with killer cell immunoglobuline-like receptor (KIR)-ligand mismatch in graft versus host (GvH) direction. Clinical data and experiments in mice indicate a beneficial influence on relapse rates, graft acceptance and Graft-versus-Host disease (GvHD). We determined the incidence of allo-reactive donor type NK cells after HLA A-, B-, DR-, DQ-matched allogeneic HSCT on a functional level. Clinical course, chimerism (PCR), immune-reconstitution (FACS) and frequencies of functional active and allo-reactive NK cells (ELISpot) were longitudinal determined in 19 patients so far. Patients (pts) suffered for high risk AML (7 pts), CML failing cytogenetic response to imatinib (3 pts), poor risk ALL (2 pts), relapse/refractory high-grade NHL (6 pts) and Multiple Myeloma (13q-) (1 pt). All patients received myeloablative conditioning regimens and GvHD-prophylaxis with cyclosporine A or tacrolimus and short course mycophenolat mofetil without in vivo or ex vivo T cell depletion. Chimerism analyses ensured hematopoietic reconstitution from donor type in 19/19 patients. In 3/19 patients NK cell activity was absent even against HLA class I negative control target cells. Absence of functional active NK cells correlates with severe acute GvHD accompanied by high doses of glucocorticosteroid medication. In all other patients we detected at least once functional active NK cells in peripheral blood. In 4/19 cases we detected allo-reactive NK cells after HSCT at days (d) +28, +68, +128 (case 19), d +56 (case 8), d +355 (case 1) and d +379 (case 13). Two cases were transplanted in KIR-ligand mismatch in GvH direction (donors HLA-CAsn80 and -CLys80, recipients missing HLA-CLys80). Allo-reactive NK cells were absent in all patients with known complete KIR-ligand match. Flow cytometry data on reconstitution of NK cell repertoire showed individual heterogeneous results. After median observation time post HSCT of 268 d (31–902) 3 patients died due to relapse. None of the patients with NK cell allo-reactivity experienced relapse. This is the first proof of circulating functionally active, allo-reactive NK cells after HLA-A, -B, -DR and -DQ matched HSCT. We detected NK cell allo-reactivity in all donor-recipient pairs with KIR-ligand (HLA-C) mismatch in GvH direction. After haplo-mismatch HSCT and T cell depletion NK cell allo-reactivity is restricted early after transplantation (within 3 months). In contrast, we detected late onset (>1 year) of NK cell allo-reactivity after one-locus (HLA-C) mismatch HSCT without T cell depletion of the grafts.
Prevention of Post Transplantation Lymphoproliferative Disorders by Adapted Management of EBV Reactivations. A Monocentric Prospective Study on 101 Patients.
Authors: Choquet Sylvain , Varnous Shaida , Deback Claire , Amiel Corinne , Golmar Jean Louis , Gandjbackhch Iradj , Raphael Martine , Leblond Veronique
Published: October 2019

Background:

Post-transplantation lymphoproliferative diseases (PTLD) represent a rare but aggressive graft complication. Patients who have received a solid organ transplantation have a 20 to 120 fold higher incidence of non-Hodgkin’s lymphoma. EBV reactivation represents a major predictive factor for PTLD, especially during the first year after transplantation, but there is no consensual attitude in this situation

Aim:

We conducted a monocentric prospective study in the Hospital of Pitie Salpêtriere, Paris, France, on all new heart or lung-heart transplanted patients. EBV viral load was systematically followed and confirmed reactivations were treated or surveyed, depending on viral load.

Methods:

101 patients were included between January 2004 and December 2006. Twelve to 15 blood samples per year were analysed. If the viral load was more than 50N, patients were treated by diminution of the immunosupression and one injection of rituximab (375 mg/m2), between 10N and 50N, only the immunosupression was modified, and rituximab was used in case of failure, and below 10N, a simple survey was decided. Correlation with CMV reactivation has been analysed.

Results:

45 (44%) patients presented an EBV reactivation. A simple survey has been sufficient in 29 cases, immunosupression decrease was the only treatment in 8 cases, 2 patients had to be treated in a second step by rituximab, because of stability or increase of the viral load beside the immnosupression modification, and 6 patients have been treated by rituximab as first treatment. All EBV reactivations have been controlled by this attitude, no PTLD has been diagnosed during this period and graft rejection rate did not change. From 1987 to December 2003, 24 PTLD have been treated in the same unit (18 EBV positive, 1 unknown), of which 13 were early PTLD (all EBV positive) with a diagnosis of less than one year after transplantation. We did not find any correlation between EBV and CMV reactivations.

Conclusion:

EBV reactivation after organ transplantation can be managed by diminution of immunosupression and/or rituximab, depending on viral load, without serious complication. A prolongation of this study and a longer follow-up are necessary to know if this attitude decreases the incidence of early EBV positive PTLD.
Bortezomib Added to the Standard Mobilization Regimen of G-CSF and High-Dose Cyclophosphamide Is a Safe and Effective Combination for a High Yield Stem Cell Collection While Promoting Further Tumor Mass Reduction in Myeloma.
Authors: Stern Jessica L. , Di Carlo Brian , Schuster Michael W. , Shore Tsiporah B. , Harpel John G. , Pearse Roger , Zafar Faiza , Dymek Joanne , Ryan Jessy , Jayabalan David , Chen-Kiang Selina , Ely Scott , Leonard John P. , Coleman Morton , Niesvizky Ruben
Published: October 2019

Standard stem cell mobilization regimens for multiple myeloma patients include G-CSF alone or in combination with high dose cyclophosphamide. Given the known in vitro and in vivo synergy between alkylating agents and proteosome inhibitors, we sought to optimize the potential for concurrent cytoreduction by adding bortezomib to the mobilization regimen. Five evaluable patients, whose prior therapy consisted of six cycles of a 21-day treatment with bortezomib/dexamethasone +/− pegylated liposomal doxorubicin, were mobilized. They received IV push bortezomib at 1.3 mg/m2 on days 1, 4, 8, and 11 in combination with high-dose cyclophosphamide at 3mg/m2 and MESNA on day 8. G-CSF was given for 10 consecutive days starting on day 9. One patient began this regimen in nCR, two were in PR, and two were in CR by urine and serum immunofixation and bone marrow evaluation. Stem cells were easily harvested from each of the five patients. The number of CD34+ cells collected far exceeded the amount normally mobilized with cyclophosphamide and/or G-CSF alone, with four out of 5 patients collected in a single day.

The two patients who began the mobilization cycle in PR continued to respond positively. Their protein levels dropped an additional 8.9 and 14.6 percent respectively during the last cycle. The patient who began mobilization in nCR achieved a CR by the end of treatment. Some expected toxicities associated with high dose cyclophosphamide and G-CSF occurred. All patients experienced grade 3 and 4 cytopenias, however, they recovered and were able to continue on to transplant. Serious adverse events of grade 3 chest pain (non-cardiac), grade 4 pneumonia, and grade 4 febrile neutropenia also occurred. Bortezomib in addition to high dose cyclophosphamide followed by G-CSF is a novel, well-tolerated and efficacious combination for stem cell mobilization in patients with multiple myeloma. This regimen not only yields a high number of stem cells within a short collection time, but may further cytoreduce disease as well.

Stem Cell Collection Patients Days Required for Collection CD34+ Stem Cells (million/kg) 1 1 21.2 2 1 47.4 3 1 22 4 1 17.9 5 4 40.6
Adoptive Donor T-Cell Infusions Are Necessary and Efficient To Induce Lasting Molecular Remissions after Highly Purified CD34+ Sibling Donor Transplants for First Chronic Phase Chronic Myeloid Leukemia - Final Report and Long-Term Follow-Up of a Phase II Study.
Authors: Elmaagacli Ahmet , Peceny Rudolf , Koldehoff Michael , Ottinger Hellmut , Trenschel Rudolf , Steckel Nina K. , Beelen Dietrich W.
Published: October 2019

Since September 1998, we prospectively studied the feasibility of transplantation using purified peripheral blood CD34+ cells from HLA-identical sibling donors in first chronic phase chronic myeloid leukemia (CML). A total of 60 patients (pts) with a median pretransplant risk score of 2 (range 1–4) has been included in this study. One patient received an unmanipulated graft due to poor CD34+ donor cell mobilization, while three pts (5%) were successfully retransplanted with an unmanipulated graft from the primary donor after secondary graft failure (n=2) or from an unrelated donor after hematologic relapse (n=1). As part of the study protocol, all pts were closely monitored for BCR-ABL transcripts using real-time RT-PCR analysis of peripheral blood cells as well as BCR-ABL-interphase FISH and metaphase karyotyping of marrow cells. Of the 60 pts, 56 were eligible for the application of donor lymphocyte infusions (DLI), but 7 pts did not receive DLI due to sustained molecular remission and complete chimerism. Thirty-one pts (52%) received DLI because of increasing BCR-ABL transcript levels or hematologic relapse, and 18 pts (30%) as programmed T-cell add-back. The median starting dose was 0.33 (0.01 – 10) x 106 CD3+ cells per kg with a median maximum dose 3.3 (0.17 – 100) x 106 CD3+ cells per kg. DLI induced a lasting reduction of median BCR-ABL transcript levels (BCR-ABL/GAPDH ratio) of more than 3 log10 and the estimate of being in a complete molecular remission at 7 years is 83% ± 5%. Six pts. (10%) did not respond to DLI, but 4 of these pts. attained a cytogenetic and molecular response by imatinib and/or interferon treatment. The cumulative risk of grades II-IV acute GvHD is 15% ± 5% for all study pts, and the risk of chronic GvHD is 25% ± 6%, respectively. After a median follow-up period of 46 (range 6 – 86) months for all pts, the cumulative 7-year survival estimate is 91% ± 4% (survival rate 92%). Causes of death were disease progression, secondary malignancy, liver failure, septicemia, and systemic capillary leak syndrome in one patient each. In conclusion, the concept of highly purified peripheral blood CD34+ cell transplantation in conjunction with adoptive DLI is associated with a particularly low risk of non-relapse mortality and allows induction of lasting molecular disease control in the majority of first chronic phase CML patients.
Non-Myeloablative Allogeneic Hematopoietic Stem Cell Transplantation in Patients with Acute Myelogenous Leukemia.
Authors: Shapira Michael Y. , Panagiotis Tsirigotis D. , Or Reuven , Resnick Igor B. , Gesundheit Benjamin , Slavin Shimon
Published: October 2019

Despite increases in CR rates, most patients with AML will relapse following successful induction treatment. Allogeneic stem cell transplantation (SCT) remains the most effective post-induction relapse preventing treatment. Similarly, for patients with relapsed/chemorefractory disease, SCT represents the only possible curative treatment modality. However, because of the significant morbidity and mortality of myeloablative conditioning regimens, SCT has been mainly restricted to young patients without co-morbidities. Because of these limitations, currently most transplant centers limit the application of SCT to patients younger than 50–55 years old.

This study included 73 patients (M=37, F=46, median age 39-years, range (17–65)) with AML treated with a single nonmyeloablative (NST) regimen in the BMT department of Hadassah University Hospital. The majority of patients had advanced AML pre-transplant (CR1 n=27; CR2 n=14; CR3 n=1; relapse n=31; chemorefractory disease n=24). All patients were prepared by the NST protocol that included IV fludarabine 30mg/m2/day (on days -10 to -5) and oral busulfan 4mg/kg/d or IV busulfex at a dose of 3.2 mg\kg\d (on days -6 and -5), with IV ATG (on days -4 and -1).

Results: Engraftment of neutrophils was achieved in 70/73 (96%) patients within a median of 15 (range 9–31) days, while engraftment of platelets was achieved in 69/73 patients (95%) within a median of 12 (range 0–86) days. Chimerism analysis data were available for 68 patients. Complete donor chimerism in the peripheral blood was achieved in 58/68 (85%) patients. 42/68 patients (62%) who survived at least one month and had a sustained engraftment, developed aGVHD. The cumulative incidence of severe aGVHD grade III-IV was 27% (18/68 patients). 35/65 (54%) patients who survived more than 100 days developed cGVHD, which was extensive in 22 (34%) cases. The cumulative incidence of NRM at 100d and 1y was 12% and 28% respectively. With a median follow up of 38m (range 10 – 118), 24/73 patients survived. Twenty-one patients survive in continuous CR, while 3 patients who relapsed post-transplant survive in CR after a second allo-SCT. The estimated long-term OS for the whole group is 30% (figure 1). 27/73 patients underwent transplantation while in CR1. Two out of these 27 patients relapsed and died, while another 8 patients died due to transplant associated complications. The estimated long-term OS for this subgroup is 63% (17/27 patients surviving in continuous CR) (figure 2).

In conclusion, the results of our study showed that our NST approach is associated with a potent anti-leukemic efficacy. The results of our study look comparable to those obtained after standard myeloablative conditioning even when stratified into disease status subgroups. Prospective randomized trial comparing myeloablative vs nonmyeloablative regimens low-risk patients is warranted.

Figure Figure Figure Figure
Four-Year Follow-Up after High-Dose Immunosuppressive Therapy (HDIT) for Severe Systemic Sclerosis (SSc).
Authors: Nash Richard A. , McSweeney Peter , Crofford Leslie , McDonagh Kevin T. , Mayes Maureen , Nelson J. Lee , Gooley Ted , Holmberg Leona , Shulman Howard , Wener Mark , McLaughlin Bernadette , Henstorf Gretchen , Molitor Jerry , Seibold James , Sullivan Keith , Furst Daniel
Published: October 2019

To determine if sustained responses could be achieved after HDIT and rescue with autologous CD34-selected hematopoietic cells, we assessed the 4-yr outcome in patients (pts) with severe SSc. Thirty-four early (≤4 years), diffuse [mod. Rodnan skin score (mRSS) ≥16] SSc pts with predefined visceral organ dysfunction (primarily lung) were included. G-CSF was used for mobilization of peripheral blood stem cells and the autologous graft was CD34-selected. HDIT included cyclophosphamide (120 mg/kg), antithymocyte globulin (90 mg/kg) and total body irradiation (800 Gy) (with lung shielding of the last 26 pts). Neutrophil and platelet counts recovered by 9 (7–13) and 11 (7–25) days, respectively. There was a significant improvement in skin (−73%, p<0.0001) and in overall function (HAQ-DI: −56%, p<0.0001) at 4 (1–8) years in evaluable pts compared to baseline (Table). Lung, heart and kidney function, in general, remained clinically stable. Importantly, biopsies confirmed a response in skin associated with resolution or a significant decrease of dermal fibrosis. Seventeen (63%) of 27 evaluable patients surviving at least 1 year after HDIT continued to have sustained responses at 4 (1–8) yrs and required no DMARD (drug-modifying antirheumatic drug) treatment. Twelve patients died (transplant-related=8 [23%]; SSc-related=4 [12%]). Two transplant-related deaths were secondary to renal crisis events that occurred within 3 months of HDIT. The other 6 transplant-related deaths were cardiac or pulmonary toxicities (n=4), EBV-PTLD (n=1) and a myelodysplastic syndrome (n=1). The estimated overall survival was 64% at 5 yrs. In conclusion, sustained responses were observed in 63% of evaluable patients after HDIT (median: 4 yrs) and marked improvement in skin and overall function was observed in high-risk SSc pts. A decrease in skin score was associated with skin remodeling.

Assess-ments Mean Baseline (BL) (n=34) Year 1–2 Difference from BL* Year 3–4 Difference from BL* Year 5–8 Difference from BL* Difference at 4 (1–8) Yrs from BL* * Difference between last measurement in that time period from baseline. mRSS (skin score) 30.12 −17.56, p<0.0001 (n=25) −21.24, p<0.0001 (n=21) −21.82, p<0.0001 (n=11) −22.08, p<0.0001 (n=25) mHAQ (disability score) 1.85 −1.26, p<0.0001 (n=23) −1.08, p<0.0001 (n=20) −1.50, p<0.0001 (n=11) −1.03, p<0.0001 (n=26) FVC (%) 71.53 4.48, p=0.06 (n=27) 2.09, p=0.56 (n=23) 10.36, p=0.007 (n=11) 2.11, p=0.50 (n=27) DLCO (%) 60.09 −1.37, p=0.60 (n=27) −3.70, p=0.31 (n=23) −2.27, p=0.51 (n=11) −7.07, p=0.02 (n=27) Creatinine (mg/dL) 0.78 0.26, p=0.02 (n=26) 0.25, p=0.009 (n=23) 0.13, p=0.008 (n=11) 0.25, p=0.003 (n=27) Ejection Fraction (%) 63.24 −0.21, p=0.90 (n=24) −2.84, p=0.06 (n=19) −3.85, p=0.03 (n=11) −2.37, p=0.06 (n=27)
Genetic Polymorphisms Correlated with Outcome in Multiple Myeloma Patients Receiving High Dose Melphalan.
Authors: Dumontet Charles , Landi Stefano , Reiman Tony , Plesa Adriana , Bellini Ilaria , Barale Roberto , Pilarski Linda , Tavtigian Sean , Gemignani Federica
Published: October 2019

High dose melphalan is an essential component in the treatment of patients with advanced multiple myeloma. However little is known regarding genetic polymorphisms possibly correlated with patient outcome in this setting. To identify polymorphisms predictive of survival and/or toxicity we performed a SNP analysis in 83 patients having received high dose melphalan for multiple myeloma. All patients had received one or two courses of high dose melphalan with stem cell reinfusion. DNA was obtained from peripheral blood mononuclear cells and analyzed for 230 SNPs in genes selected for their role in drug metabolism, DNA repair, cell cycling or apoptosis. There were 48 males and 35 females and the median age was 58 years. The average number of VAD courses received before high dose melphalan was 4 (3–6). 49 patients received two high dose melphalan courses. The best response to melphalan was CR in 22 pts, very good response in 14 pts, partial response in 37 patients and stable or progressive disease in 10 patients. 37 patients had experienced disease progression and 8 patients had died at last follow-up. 29 patients experienced high grade (III-IV) mucositis after high dose melphalan. The median freedom from progression was 40 months. CYP2E1 polymorphism 7632 was correlated with freedom from progression (FFP) after high dose melphalan (p=0.02). SULT1A2 704 polymorphism was correlated both with FFP (p=0.002) and with overall survival (p=0.001). Occurrence of severe mucositis after high dose melphalan therapy was correlated with XRCC1 polymorphism 1198 (24% vs 51% of high grade mucositis) as well as ATM polymorphisms. These results suggest that SNPs of genes involved in drug metabolism or repair could be used to distinguish patients subgroups with different toxicity/efficacy profiles after high dose melphalan.

OS according to SULT1A2 N235T OS according to SULT1A2 N235T
Increased Calreticulin Expression in Clonal Plasma Cells Is Associated with Complete Response to Melphalan and Autologous Stem Cell Transplant in Systemic Light-Chain Amyloidosis.
Authors: Zhou Ping , Olshen Adam B. , Teruya-Feldstein Julie , Comenzo Raymond
Published: October 2019

Systemic light-chain amyloidosis (AL) is a protein deposition disorder and a monoclonal plasma cell disease. Treatment of AL has focused on the reduction or elimination of the clonal plasma cells that make amyloid-forming light chains. High-dose melphalan and stem cell transplant (MEL SCT) is an effective therapy for selected AL patients. At 3 months post-SCT, response of the clonal plasma cell disease can be reliably scored; the achievement of a complete response (CR= immunofixation negative) is usually associated with extended survival while no response (NR= < 50% reduction) is not. Many factors likely contribute to this distribution of responses. In order to identify factors specific to clonal plasma cells, gene expression profiles (GEP; Affymetrix U133 PLUS 2.0) were obtained using FACS-sorted CD138+/DAPI- plasma cells (>95% pure) from untreated AL patients before SCT. Supervised analyses were then performed based on responses at 3 months post-SCT, comparing the CR (n=4) and NR (n=5) groups. The basic analysis was a t-test, filtering genes for differential expression at p<0.01, and those that passed were subject to EASE analysis in order to identify gene ontology families over-represented on the gene list. A secondary filtering selected only genes with two-fold difference in expression between CR and NR, average expression of at least 1000, and inclusion in an EASE group that had a p<0.05. Five genes were identified as over-expressed in the CR group: WARS, SHMT2, IARS, CALR, and PSMB4. Our initial validation studies, using purified baseline clonal plasma cells from 11 patients and baseline marrow biopsy specimens from 25 patients, have focused on calreticulin (CALR) and tryptophanyl tRNA synthetase (WARS). By real-time polymerase chain reaction studies with GAPDH and normal plasma cell controls, the expression levels of CALR were significantly lower in NR patients (p=0.02) while differences in WARS expression did not achieve significance (p=0.25). By immunohistochemical (IHC) staining for CALR in marrow biopsies, a CALR IHC index was developed, with scoring of the fraction of plasma cells expressing CALR (over total CD138 positivity) and intensity of CALR staining (both scored 0, 1, 2 or 3; the scores were multiplied to compute the index, range 0–9). CALR-stained marrows were scored blindly. Patients with CALR index of 9 had an 88% likelihood of achieving CR while those with scores of 0–6 had a 39% chance (Fisher’s exact test, p=0.046). In sum, calreticulin expression in clonal plasma cells from AL patients is associated with the response to MEL SCT. Further studies are on-going to determine the basis for this association.
Epitope-Specific Typing (EST) for HLA-DPB1 Matching: Proof of Principle for an Innovative Approach to Unrelated Hematopoietic Stem Cell Donor Selection.
Authors: Zino Elisabetta , Vago Luca , Di Terlizzi Simona , Mazzi Benedetta , Amato Luigi D’ , Rossini Silvano , Ciceri Fabio , Roncarolo Maria G. , Bordignon Claudio , Fleischhauer Katharina
Published: October 2019

Background. Conventional matching of patients and their unrelated donor (UD) hematopoietic stem cells (HSC) by 4-digit molecular HLA typing is associated with lengthy donor searches and elevated social costs. 80% of UD transplants are performed across DPB1 mismatches which, if involving disparity in host versus graft (HvG) direction for an immunogenic T cell epitope, have been shown to be associated with poor clinical outcome of transplantation for hematopoietic malignancies and beta-thalassemia. In this study we have developed an innovative approach of DPB1 epitope- rather than allele-specific matching, by only two PCR reactions (epitope-specific typing; EST). Moreover, we have determined allelic DPB1 frequencies in Italy, confronted them with the ones previously reported for other ethnic groups, and calculated the probability of finding non-permissive DPB1 mismatches in unrelated HSC donor searches.

Methods. High resolution genomic DPB1 typing and EST were performed in parallel on blood samples taken from 112 healthy unrelated Italian blood donors.

Results. EST of DPB1 alleles encoding the immunogenic T cell epitope yielded 100% concordant results with high resolution DPB1 typing in all 112 samples studied, and is therefore suitable to univocally determine the presence or absence of non-permissive DPB1 disparities. The overall frequency of DPB1 alleles encoding the shared T cell epitope in the Italian population was 23.15%. Importantly, we show that based on DPB1 allelic polymorphism in the four ethnic groups representative of the world-wide UD registries, over 75% of UD matched for the other HLA loci will not present a DPB1 epitope disparity in HvG direction, demonstrating that prospective UD-recipient DPB1 matching by EST does not significantly limit the number of suitable donors, and has a negligible impact on the time and cost of the search.

Conclusions. EST is a challenging alternative to conventional tissue typing which, if applied more broadly to HLA loci other than DPB1, could fundamentally change current approaches to UD searches.
Transplant-Related Mortality in Patients ≥50 Years of Age Is No Lower after Transplantation from Young Fully Matched Unrelated Donors (MUD) Than from Matched Siblings When All Patients Are Given the FLUBUP Regimen with Thymoglobulin.
Authors: Russell James A. , Chaudhry M. Ahsan , Quinlan Diana M. , Turner A. Robert , Larratt Loree M. , Geddes Michelle , Storek Jan , Savoie Mary Lynn , Brown Christopher , Bahlis Nizar J. , Stewart Douglas A.
Published: October 2019

There are reports indicating that older patients (pts) may have better outcomes after stem cell transplantation (SCT) from young fully matched unrelated donors (MUD) than from matched siblings (MRD) perhaps because of donor age. In some of these studies MUD and MRD recipients may have been treated differently, for example with more intense GVHD prophyaxis for MUD SCT such as antithymocyte globulin (ATG). We have compared outcomes of SCT pts receiving myeloablative fludarabine & busulfan based conditioning (FLUBUP) between 05/99 and 05/05. Only 10/10 (HLA–A, −B, C, DR & DQ) matched SCT were considered. Recipients of MRD SCT were ≥50 years old, MUD SCT recipients were unselected for age but their donors were ≤30 years old. All pts received fludarabine 50mg/m2 on days −6 to −2 and IV busulfan (Busulfex, PDL Pharma) at a myeloablative dose of 3.2 mg/kg once daily days −5 to −2 inclusive (FLUBUP) +/− TBI 200cGy x 2 on day −1 or 0. Prophylaxis for GVHD was cyclosporine A, methotrexate with folinic acid and Thymoglobulin (Genzyme) 4.5 mg/kg in divided doses over 3 consecutive days pretransplant finishing D0.[table 1]These data demonstrate that TRM for older SCT pts given the FLUBUP regimen with ATG is similar to that for adults in general transplanted from young MUD. When using these protocols in older pts there is usually no justification for the expense and inconvenience of using a young MUD in preference to a matched sibling.

Patient, donor & SCT characteristics and outcomes Unrelated Related p * at 5 years Number 41 63 Patient age median (range) 44 (16–61) 55 (50–65) <0.0001 Donor age median (range) 24 (19–30) 52 (37–71) <0.0001 Low risk (Acute leukemia CR1/2, CML CP1) 22 (54%) 14 (22%) 0.0015 CMV+ve recipient or donor 31 (76%) 53 (84%) ns Female to male SCT 8 (20%) 18 (29%) ns Blood cells 20 (49%) 56 (89%) <0.0001 CD 34+ cell dose x 10e6 median (range) 5.35 (0.91–15.47) 4.23 (0.83–13.52) 0.08 TBI (not TRM risk factor) 19 (46%) 10 (16%) 0.0014 Acute GVHD II–IV 21±7% 16±5% ns Acute GVHD III–IV 10±5% 5±3% ns Chronic GVHD 64±7% 61±9% ns Pts≥50 TRM * 26±15% (n = 12) 16±5% ns Low risk TRM 5±5% 0% ns High risk TRM 31±12% 23±7% ns BCT TRM 10±7% 19±6% ns All pts TRM 16±6% 16±5% ns
TNFR25 Expression on CD4+CD25+ T Cells: Down Modulation of Regulatory Activity.
Authors: Deyev Vadim , Roskos Melinda , Levy Robert B. , Podack Eckhard R.
Published: October 2019

TNFR25 (“DR3”) is a member of the TNF receptor family that is expressed by activated CD4+ and CD8+ T cells. To determine if activated CD4+CD25+ T cells also expressed this TNFR family molecule, B6 CD4+CD25+ T cells were stimulated with anti-CD3/CD28 coated beads (kind gift of Dr. B. Blazar, U. Minn.) and expanded for 3–4 days. TNFR25 expression was readily detected on CD4+CD25+ FoxP3+ T cells. Since other members of the TNF receptor family (GITR, OX40, 4–1BB) are known to influence T regulatory cell function, we investigated whether TNFR25 signaling can regulate CD4+CD25+ T cell activity. TNFR25 triggering in B6-wt T regulatory CD4+CD25+ cells with the recombinant TNFR25 ligand TL1A or agonistic anti-TNFR25 antibody (4C12) resulted in reduction of their ability to suppress anti-CD3 induced ex-vivo proliferation of CD4+CD25− cells. 4C12 mediated TNFR25 signaling also reduced B6-wt Treg mediated inhibition of peptide induced proliferation of OVA-specific B6 CD8+ (OT-I) cells. To further investigate a role for TNFR25 in Treg cell regulation, TNFR25 (full length) transgenic mice were generated and bred onto the BL/6 background. CD4+CD25+ cells from these TNFR25 tg mice were found to possess diminished T regulatory activity in vitro as determined by their diminished inability to regulate proliferation by B6-wt CD4+ and OT-I CD8+ T cells. To assess their in vivo regulatory activity, B6-wt and B6 TNFR25 tg Treg cells were examined for their ability to inhibit graft vs. host disease (GVHD) following allogeneic MHC class I/II mismatched BMT. In contrast to B6-wt Treg cells, TNFR25 tg Treg cells exhibited significantly diminished ability to regulate the onset of GVHD in vivo as assessed by weight loss and clinical symptoms. Using agonistic antibody, stimulation of TNFR25 on transgenic Treg cells was also found to effectively remove the ex-vivo regulatory activity expressed by this population. To exclude any possible direct co-stimulatory effects of 4C12 antibody on the responding proliferating cells, CD4+CD25−T cells from TNFR25 dominant negative transgenic mice were employed. 4C12 mab again abolished Treg cell inhibitory activity. The effect of TNFR25 agonists on T reg cell activity in vivo is being further investigated in both mouse models of GVHD and IBD diseases. Initial observations administering 4C12 post-allogeneic BMT together with B6-wt Treg cells indicate a reduction in their ability to regulate GVHD. In total, these studies identify TNFR25 as a new potential target for augmenting CD4+ and CD8+ responses by concomitant direct co-stimulation of effecter cells and inhibition of T regulatory cell function.
Busulfan Selectively Induces Cellular Senescence Via a p53-Independent but Erk-p38 MAPK-Dependent Mechanism.
Authors: Probin Virginia , Bai Aiping , Zhou Daohong , Wang Yong
Published: October 2019

Busulfan (BU), an alkylating agent, has been used extensively for the depletion of leukemia cells and normal hematopoietic stem cells (HSCs) prior to bone marrow transplantation. However, its mechanism(s) of action is unknown. Our laboratory has previously shown that BU primarily depletes HSCs by induction of senescence, but not apoptosis. In the present study, we investigated the molecular mechanisms whereby BU induces cellular senescence utilizing WI38 human diploid fibroblasts as a model system. We found that WI38 fibroblasts incubated with BU (from 7.5 to 120μM) for 24 h underwent senescence but not apoptosis in a dose-independent manner, whereas cells incubated with 80μM and 20μM etoposide (Etop) committed to apoptosis and senescence, respectively. The induction of WI38 cell senescence by Etop was associated with p53 activation and could be attenuated by down-regulation of p53 using a-PFT or p53 siRNA. In contrast, WI38 cell senescence induced by BU was associated with prolonged activation of Erk, p38 and JNK, and could be suppressed by the inhibition of Erk and p38 MAPKs with PD98059 and SB203580, respectively. Upon release from Erk and p38 inhibition, BU-treated cells proceeded to DNA synthesis and cell division. However, inhibition of p53 with a-PFT or p53 siRNA, or JNK with SP600125, failed to protect WI38 cells from BU-induced senescence. These findings suggest that BU is a distinctive chemotherapeutic agent that can selectively induce cellular senescence through the Erk and p38 MAPK pathways.
Generation and Functional Characterization of T Cells Against Candida albicans as Potential Immunotherapy after Allogeneic Stem Cell Transplantation.
Authors: Lehrnbecher Thomas , Beck Olaf , Koehl Ulrike , Roeger Frauke , Hunfeld Klaus-Peter , Latge Jean Paul , Sarfati Jaqueline , Klingebiel Thomas , Tramsen Lars
Published: October 2019

Invasive fungal infections (IFI), in particular infections due to Aspergillus spp and Candida spp, still pose considerable problems in patients undergoing allogeneic stem cell transplantation (SCT). Despite the availability of new antifungal agents, morbidity and mortality of IFI are still unacceptable high. Although neutropenia is known as the single most important risk factor for IFI, there is a growing body of evidence that T cells play a major role in the defense against fungi. Therefore, adoptive immunotherapy with T cells against Candida spp. might be an interesting therapeutic option in patients undergoing allogeneic SCT. After overnight incubation of 1×108 peripheral blood mononuclear cells from 4 healthy individuals with cellular extracts of C.albicans, activated T cells were selected using the IFN-γ secretion-assay (Miltenyi Biotec, Bergisch Gladbach, Germany). After 14 days of culture, T cell clones were generated by limiting dilution and incubated for another 14 days. The median number of cells obtained was 2.6×107 (range, 0.85–5.75×107). Flow cytometry revealed a highly homogenous population of CD3+CD4+ cells (97.2% ± 2.6; n=6), of which an average of 8.6% (range, 4.8–58.2%) produced IFN-gamma on re-stimulation with C.albicans antigens, as assessed by intracellular cytokine staining assay. 20.5% (range, 5.8–72.4%) of the generated cells produced TNF-alpha, whereas no significant number of cells produced TH2 cytokines such as IL-4 and IL-10, indicating that the generated T cell clones were TH1 cells. The percentage of IFN-gamma producing T cells was significant upon stimulation with C.albicans and C.tropicalis, whereas less than 1% of cells produced IFN-gamma upon stimulation with antigens of other yeasts such as C.glabrata, Debaryomyces hansenii and Kluyveromyces lactis and molds such as A.fumigatus, Penicillium chrysogenum and Alternaria alternata. Compared to CD4+ T cells of the original fraction, the isolated and expanded anti-Candida T cells showed reduced alloreactivity, as assessed by means of CSFE. In addition, a strong proliferation of the generated anti-Candida T cells was seen after re-stimulation with C.albicans antigens. The potency of the generated T-cells to damage C.albicans was evaluated using the XTT assay. Compared to polymorphonucelar cells (PMNs), APCs and T-cells alone or to the combination of PMNs with T cells or APCs, respectively, the combination of PMNs, APCs and T-cells showed highest fungal damage (n=4).

In conclusion, our data suggest that the isolation and expansion of anti-Candida T cells is possible and feasible. The generated T cells show low alloreactivity in vitro and increase the antimycotic potential of phagocytes. Thus, antimycotic T cells might become an important tool in the prophylaxis and therapy of IFI in patients after allogeneic SCT.
Association of TLR-4 Mutations in Host and Donor Cells and the Risk for Acute GVHD after Allogeneic Hematopoietic Stem Cell Transplantation.
Authors: Imado Takehito , Iwasaki Tsuyoshi , Kuroiwa Takanori , Kitano Sachie , Sano Hajime , Ogawa Hiroyasu
Published: October 2019

Lipopolysaccharide (LPS) has been implicated in the pathogenesis of acute graft-versus-host disease (GVHD). The toll-like receptor (TLR)-4 has been recently identified as a major receptor for LPS. It is possible that mutations of TLR-4 have been associated with LPS hyporesponsiveness, thereby reducing the risk of acute GVHD in allogeneic hematopoietic stem cell transplantation (HSCT). However, stimulation of TLRs may also initiates a signaling cascade for induction of various pro-inflammatory cytokines, leading to microbial eradication. Therefore, mutations of TLR-4 may increase risk of microbial infection, thereby accelerating acute GVHD in allogeneic HSCT recipients. Retrospective studies to determine the frequency of TLR-4 mutations and their possible association with acute GVHD contradict each other. In the present study, we examined the association of TLR-4 mutations in host and donor cells and the risk for acute GVHD after allogeneic HSCT. To examine the role of TLR-4 in host cells, we analyzed TLR-4 expression in the intestine by immunohistochemistry. Lethally irradiated C3H/HeN mice received allogeneic HSCT from C57BL/6 donors. TLR-4 expression in the intestinal epithelial cells was significantly increased after GVHD induction. Next, we used a mouse strain (C3H/HeJ) that has a single amino acid substitution in its TLR-4 as recipients in an allogeneic HSCT. Lethally irradiated either C3H/HeN or C3H/HeJ hosts received HSCT from C57BL/6 donors. C3H/HeN recipients receiving C57BL/6 donors developed significantly less GVHD as measured by mortality and intestinal histopathology compared with C3H/HeJ recipients receiving C57BL/6 donors. TNF-a mRNA expression in C3H/HeJ recipients was stronger in the intestine but weaker in the spleen and the liver comparing to that in C3H/HeN recipients. These results suggest that TLR-4 mutation in host cells reduces the expression of pro-inflammatory cytokines in the spleen and the liver, while it also reduces microbial eradication in the intestine, increasing the risk of LPS stimulation of macrophages in the intestine, thereby augmenting TNF-a expression and damages of the intestine. Next, we examined the role of TLR-4 in donor cells. Lethally irradiated C57BL/6 hosts received allogeneic HSCT from either C3H/HeN or C3H/HeJ donors. Mice receiving C3H/HeJ donors developed significantly less GVHD as measured by mortality and intestinal histopathology compared with mice receiving C3H/HeN donors. Taken together, these results suggest that TLR-4 in host cells is crucial in the microbial eradication in the intestine and the protection of intestinal GVHD, while responsiveness of donor cells to LPS may be an important risk factor for acute GVHD.
Effect of Aging on Host Dendritic Cell (DC) Numbers and Function, and Response to Regulatory DC Treatment in a Murine Allogeneic Transplant Model.
Authors: Schlueter Annette J. , Park Joon Seong , Wilke Werner W. , Fan Ji , Gingrich Roger D.
Published: October 2019

Graft versus host disease (GVH) incidence and severity increases with older patient age. Host dendritic cells (DC) are responsible for initiating GVH, presumably as a result of activation at the time of transplant conditioning (as they are short lived after transplantation). Age-related alterations in host DC function have not been studied in a transplant setting. In this study, young (8–14 wk) and older (12–16 mo) mice were treated with the conditioning regimen of lethal irradiation (1100 cGy) and assessed at 6 h later for changes in DC costimulatory molecule expression and cytokine secretion. Older mice had slightly higher numbers of splenic classical DC (cDC; CD11chi Cl II+ B220−) and plasmacytoid DC (pDC; CD11clo Cl IIlo B220+) both before and after irradiation. No differences were observed in CD40, CD80, or CD86 expression between young and older mice, either before or after irradiation. IL-12, IFNγ, and TNFα producing cDC were more numerous in young untreated mice than in older mice. However, these cells decreased in young mice and increased in older mice after irradiation, resulting in generally higher numbers of cells producing these cytokines in older mice. IL-12, IFNγ, and TNFα producing pDC were more frequent in older mice than in young mice prior to irradiation, and both older and young mice showed increased frequency of cells producing these cytokines after irradiation. Overall, the highest numbers of cDC and pDC producing IL-12, IFNγ, and TNFα were present in older mice after irradiation. In both cDC and pDC populations, older mice had a higher frequency of IL-10+ cells prior to irradiation. After irradiation, young mice had a higher frequency of IL-10+ cells. These findings indicate that the balance of cytokine production in older mice after conditioning with irradiation is shifted to immunostimulatory cytokines rather than immunosuppressive cytokines. Murine GVH can be prevented by treatment with regulatory DC (rDC) in young animals (Sato et al., Immunity 2003). To determine whether this treatment is feasible in older animals, older BALB/c mice were transplanted with young C57Bl/6 BM. A subset of each group received age-matched host derived rDC. As expected, among mice that did not receive rDC, old recipients got more rapid and severe GVH than young recipients. This correlated with an increased frequency of residual host DC at the time of GVH in the older group. rDC were used to successfully treat GVH in older (as well as young) transplant recipients. These results provide the foundation for a cell based therapeutic approach to the treatment of GVH in older individuals.