bluuebunny/crossref_metadata_2025 huggingface dataset:
An Intact Spleen Is Required for Alloimmunization to Transfused Red Blood Cells Due to Intrasplenic Activation of CD4+ T Cells.
Authors: Hendrickson Jeanne E. , Roback John D. , Hillyer Christopher D. , Zimring James C.
Published: October 2019

Background: Factors influencing rates of alloimmunization to antigens on transfused red blood cells (RBC alloimmunization) are poorly defined. In particular, the role of the spleen with respect to alloantibody formation is unclear, with conflicting clinical reports in the literature. Moreover, the complexities of multiply mismatched antigens and antigen priming due to transfusions prior to splenectomy make human studies difficult to interpret. To better define the role of the spleen in RBC alloimmunization, we utilized a murine model of transfusion medicine (with the model RBC antigen mHEL (membrane bound hen egg lysozyme)).

Methods: Cohorts of splenectomized and non-splenectomized mice (C57BL/6 × B10.BR) were transfused with the human dose equivalent of 1 unit of leukoreduced mHEL RBCs. RBC alloimmunization was assessed by anti-HEL IgG specific ELISA. The role of antigen-specific CD4+ T cells was studied by the adoptive transfer of 1.5 × 106 HEL-specific CFSE-labeled CD4+ T cells from 3A9 TCR transgenic donors. Adoptively transferred cells were visualized using a congenic marker (Thy1.1); enumeration and division of these cells were monitored by flow cytometry in liver, spleen (if applicable), and lymph node preparations.

Results: Splenectomy dramatically decreased RBC alloimmunization; 14 of 14 splenectomized mice (from 3 experiments) had undetectable to very low levels of anti-HEL IgG following transfusion with mHEL RBCs (average 6.3 fold less than non-splenectomized mice, 95% C.I. 4.6). Moreover, ten of ten splenectomized mice failed to make detectable levels of anti-HEL IgG even following the adoptive transfer of HEL-specific CD4+ T cells. In comparison, elevating the precursor frequency of HEL-specific CD4+ T cells increased RBC alloimmunization by 10,000 fold in non-splenectomized mice. Proliferation and division of CD4+ T cells were detectable in both spleen and liver preparations of non-splenectomized mice by day 3 following transfusion; in contrast, no expansion nor division of CD4+ T cells was seen in liver nor lymphatic preparations of splenectomized animals.

Conclusions: The low level of RBC alloimmunization seen in non-splenectomized mice in this system is limited by existing CD4+ helper T cell responses, as increasing the naive helper T cell precursor frequency dramatically increased RBC alloimmunization. Furthermore, the spleen itself is critical to CD4+ helper T cell function during alloimmunization given that, in splenectomized mice, adoptively transferred HEL-specific CD4+ T cells fail to expand, divide, or stimulate production of detectable alloantibody. Ongoing studies are investigating the phenotype of HEL-specific CD4+ T cells (as effector cells, anergic cells, or regulatory cells) in splenectomized and non-splenectomized mice. These studies have implications for preventing alloimmunization in transfusion-dependent patient populations.
Multicenter Phase II Trial of Patients with Refractory or Recurrent Multiple Myeloma with Oral Treatment of Thalidomide Combined with Oral Cyclophosphamide, Idarubicin and Dexamethasone.
Authors: Glasmacher Axel , Moehler Thomas , Goldschmidt Hartmut , Geer Thomas , Kleinschmidt Rolf , Gaska Tobias , Uwe Janssens , Schubert Jorg , Bruck Patrick , Clemens Michael R. , Bair Wolfgang , Seipelt Gernot , Schleuning Michael , Hahn-Ast Corinna , Herz Susanne , von Lilienfeld-Toal Marie , Naumann Ralf , Gorschluter Marcus , Schmidt-Wolf Ingo G.H.
Published: October 2019

Introduction and rationale: The aim of our study is to evaluate the role of thalidomide in combination with oral CID therapy in order to improve treatment wit an all oral regime.

Patients and methods: Patients with refractory or relapsed myeloma were included into this trial. Concerning inclusion criteria, 10 patients had progressive disease after standard chemotherapy, 12 no change after standard chemotherapy and required further therapy, 6 recurrent disease after standard therapy and 22 recurrence after high dose chemotherapy. At start of the treatment patients were in stages IIA (16 patients), IIB (1), IIIA (34) and IIIB (1). Idarubicin was given in capsules at 8–10 mg/m2/day for days 1–4, 40 mg dexamethasone were given on days 1–4 and 15–18, cyclophosphamide at 200 mg/m2/d d1–4 and thalidomide 100mg daily with scheduled increase to 400mg. Treatment cycles were repeated every 28 days. 3–8 cycles were applied. Supportive therapy consisted of PEG-filgrastim, cotrimoxazol, dalteparin and ibandronate. In addition, after the induction phase patients were randomized between thalidomide and thalidomide / idarubicin maintenance treatment.

Results: At this interim analysis, 66 patients have been entered into the ongoing trial; 6 patients had to be excluded for violation of unclusion criteria. Of the 60 included patients, 52 patients have been documented so far and are evaluable for toxicity. In total, 187 cycles have been applied. Median cycle number was 3 (1–8). There was one death during treatment due to progressive myeloma at cycle 1. The median age was 62 years (interquartile range 57–67). The following toxicities were observed: FUO (2 patients), pneumonia (4), pulmonary embolism (1), exsiccosis after diarrhea (1), constipation (1), sepsis (1), severe nausea (2), syncope (1), collaps after bleeding (1, not related to TCID treatment) and mucositis grade 4 (1). Hematologic toxicity grade IV was reported in 35% of the patients, mainly in cycles 1 and 2. Concerning response to treatment 41 patients are evaluable for response so far. Of these 31 (76%) achieved a PR (24 patients) or MR (7 patients) according to EBMT criteria, 4 patients a stable disease and 3 patients a PD. Three patients were not evaluable and scorded as failures.

Conclusions: T-CID is feasible and highly effective in relapsed or refractory myeloma patients. Toxicity seems to be acceptable and mainly hematologic and infectiologic. The remission rate of 76% is encouraging.
Combination Therapy with Lenalidomide, Bortezomib, Liposomal Doxorubicin and Dexamethasone (LBlipDD) May Overcome Resistance to Prior Treatment with Doxorubicin, Lenalidomide, and Bortezomib in High-Risk Multiple Myeloma (MM).
Authors: Ruckser Reinhard , Tatzreiter Georg , Kitzweger Elvira , Strecker Karin , Hraby Stefan , Buxhofer Veronika , Stampfl Margarete , Kier Peter , Habertheuer Karl H. , Hejtman Milos , Hinterberger Wolfgang
Published: October 2019

Introduction: Lenalidomide (Revlimid®) plus dexamethasone therapy and single-agent bortezomib therapy are approved for the treatment of relapsed/refractory multiple myeloma (MM) patients. A recent phase II trial has shown activity of lenalidomide/bortezomib/dexamethasone in patients with relapsed/refractory MM (Richardson, IMW 2007). Here, we present a case report on the efficacy of combination therapy with lenalidomide, bortezomib, liposomal doxorubicin, and dexamethasone in a patient who was refractory to prior treatments with bortezomib, lenalidomide, and doxorubicin.

Methods and Results: A male patient (58 years) presented with IgG-lambda MM in June 2006. Laboratory tests at diagnosis showed total protein of 123g/L (normal value, 66–87 g/L) and a serum IgG of 87.3g/l (normal value, 7–16 g/L). The patient had t(11;14)(q13;q32) translocation and a del13q14, and bone marrow aspirate showed >90% plasma cells. From July 2006 to March 2007, the patient received 3 different chemotherapy treatment regimens (VAD: vincristine, Adriamycin® [doxorubicin], dexamethasone; VDD: Velcade® [bortezomib], doxorubicin, dexamethasone; and LD: lenalidomide, dexamethasone). He showed primary resistance to VAD treatment and developed resistance after 3 and 5 cycles of VDD and LD, respectively. At that point, the patient’s free light chain (fLCh) concentration was 2,320 mg/L (normal value, 5.7–26.3 mg/L). We changed the patient’s treatment regimen to the 4-fold combination of lenalidomide plus bortezomib plus liposomal doxorubicin (lipD) plus dexamethasone (LBlipDD), (lenalidomide 25 mg day 1–21, bortezomib 1mg/m2 day 1+4+8+11, lipD 50 mg/m2 day 4, dexamethasone 40 mg day 1+2+4+5+8+9+11+12; q28 days). The patient received 3 cycles of LBlipDD from May 2007 to July 2007. This treatment combination was well tolerated with no WHO grade 3 or 4 adverse events. The patient was reassessed after 3 treatment cycles. FISH showed a complete eradication of the former cytogenetic abnormalities, bone marrow aspirate showed <5% plasma cells and serum analysis demonstrated a normalized serum IgG value, and a decrease in the fLCh from 2,320 to 173 mg/L. The patient is for the first time transfusion independent and in very good clinical condition. High-dose melphalan with autologous stem cell support is currently planned.

Conclusion: Treatment with LBlipDD leads to a good remission in a VAD-, VDD- and LD-resistant patient. The present observation suggests that the use of 4-fold combination of lenalidomide, bortezomib, liposomal doxorubicin, and dexamethasone can be effective in high-risk MM patients and warrants further investigation.
Combination Therapy of Bortezomib with Bendamustin in Elderly Patients with Advanced Multiple Myeloma. Clinical Observation.
Authors: Hrusovsky Irena , Heidtmann Hans-Heinrich
Published: October 2019

Bortezomib(Bo) was approved for therapy of relapsed Multiple Myeloma (MM) in 2003-for use in Germany 2004. There is growing evidence that combination of Bo with conventional chemotharapy agents could improve MM patients’ (pts) outcome(P.Richardson ASCO 2004). Bendamustin(Ben) is widely used for MM chemotherapy in Germany. In weekly low dose regimen it is well tolerated and highly effective in therapy of low grade lymphoma in elderly (K.Bremer ASCO2003, Abstract 2410). Ben has no cross resistances with other cytostatic drugs used in MM therapy, in low dose regimen it is well tolerated, has low bone marrow toxicity and no renal toxicity. We reported results of the combination therapy of the first 17 pts 2 years ago. Because of very good tolerability and high response rates we established the combination in our institution as a salvage-therapy for pts with relapsed MM after at least 2 previous chemotherapies. Till now we treated 40 pts - all white European-21 women, 19 men- median age 66 years (range 51–86). All pts had relapsed MM with clinical symptoms such as anemia, bone pain, progressive bone disease, several of the pts had renal failure (1 on hemodialysis). Previous therapies - median 4-(range 2–10)- included Melphalan, Dexamethason, VAD and sim. combinations, Cyclophosphamide, Bendamustin, Thalidomide, Bortezomib mono and in 3 pts tandem HD Melphalan. Therapy-regimen: Bortezomib 1–1,3 m/sqm d 1,4,8,11, Bendamustin 60mg/sqm d 1,8, Dexamethason 3x8mmg p.o. d 1–3 and 8–10 if tolerated, Ondansetron 8 mg i.v. d 1,4,8,11- q3w until best response- median number of cycles 4 (range 2–6). Early responses with symptom relief at begin of second cycle were frequently observed. Results (outcome according to SWOG criteria): ORR 85%, very good PR 25%(normal electrophoresis, normal level of free light chains in serum and urine), PR 47,5%, MR 12,5%. Remission duration in pts with at least PR- 8 Months (range 2–26 months)-19 pts are still alive. Toxicity: comprised fatigue and mostly mild thrombocytopenia without bleeding, reversible within 1 week. 8 pts had neuropathy and required symptomatic therapy with Gabapentine- most of them pretreated with Thalidomide. 9 pts had herpes zoster before aciclovir 3x400 mg daily was administered as prophylactic therapy.

Conclusion: The therapy with combination Bortezomib- Bendamustin is highly effective, safe and well tolerated. The therapy is feasible in out-patient setting. The response rates suggest that the drugs act at least additive. We think therefore that further studies are warranted.
CD4+ T Cell Lines Reactive with Acute Myeloid Leukemia (AML) Cells Generated by Competitive Limiting Dilution Culture of AML Mononulear Cells (MNC) Eliminate Autologous AML by Apoptosis Induction.
Authors: Zhong Rui-kun , Lane Thomas A. , Ball Edward D.
Published: October 2019

Traditionally, the focus of anti-tumor immunity has been on CD8+ CTL responses. It is now realized that CD4+ T cells play a relevant role in protective anti-tumor responses. HLA Class II molecules are expressed on AML blasts, predicting that AML cells may stimulate CD4+ T cells. Based on our studies of inducing AML dendritic cell (AMLDC) differentiation and priming in situ AML-reactive T cells, we developed a novel method of generating multiple autologous AML reactive T cell lines by competitive limiting dilution (LD) AML MNC culture. Most autologous AML reactive T cell lines generated from this culture were CD4+. These CD4+ T cell lines with high IFN-gamma secretion in response to autologous AML cells showed low to moderate specific lysis of autologous AML cells in 4-hour 51Cr release assays. However, co-culture assays demonstrated that these CD4+ AML reactive T cell lines exerted intense cytotoxicity toward autologous AML cells, depleting more than 95% of autologous AML line cells in 2 days, and more than 99% in 7 days, with no or weak toxicity to allogeneic AML cells and cell lines, HL60, NB4, U937 KG1a and ak LCL cell line. Anti-HLA class II, Dr, Dp, Dq mAb (67±16% inhibition) and anti-HLA-Dr mAb (69±11% inhibition) but not anti-HLA class I mAb significantly inhibited IFN-gamma secretion of six CD4+ T cell lines stimulated by autologous AML cells confirmed the HLA class II restriction of reactivity of these CD4+ T cell lines. Flow cytometry analysis showed that these CD4+ T cell lines induced significant Annexin-V expression on autologous AML cells. The induction of Annexin-V+ on AML cells by CD4+ T cell lines correlated significantly with IFN-gamma secretion in response to autologous AML cells (n=11; r=0.84). This result suggested that the most important mechanism of autologous AML cell elimination by CD4+ AML reactive T cells generated from LD-AML-MNC cultures was apoptosis induction related to IFN-gamma secretion.
CD38 Gene Polymorphisms Contribute to Genetic Predisposition to B-Cell Chronic Lymphocytic Leukemia.
Authors: Jamroziak Krzysztof , Szemraj Zofia , Grzybowska-Izydorczyk Olga , Szemraj Janusz , Cebula Barbara , Balcerczak Ewa , Wawrzyniak Ewa , Mirowski Marek , Robak Tadeusz
Published: October 2019

Genetic background and abnormal immune response are supposed to play a role in the pathogenesis of B-cell chronic lymphocytic leukaemia (B-CLL). CD38 is a leukocyte activation antigen and ectoenzyme involved in numerous immune functions as well as important prognostic factor in B-CLL. The aim of this study was to investigate potential association of two single nucleotide polymorphism (SNPs) located in CD38 gene at position 182 (C/G) in intron 1 and at position 418 (C/T) in exon 3 with genetic susceptibility to B-CLL. Furthermore, we wanted to verify whether these SNPs influence CD38 expression or clinical outcome of B-CLL patients. Genotyping of 365 Caucasian individuals (163 B-CLL patients and 202 healthy controls) was performed by restriction fragment length polymorphism (RFLP) and allele-specific polymerase chain reaction (ASPCR) assays. CD38 gene expression was quantified by real-time RT-PCR and CD38 protein expression was measured by flow cytometry in circulating B-CLL cells from 50 patients and normal B and T lymphocytes from 30 healthy controls. We found that allele frequency and distribution for both CD38 SNPs differed significantly in patients and controls with increased prevalence of alternative alleles detected in B-CLL (allele 182G found in 34.7% B-CLL patients vs. 13.4% controls, p<0.0001, and allele 418T found in 4.6% B-CLL patients vs. 0.5% controls, p=0.0002). Logistic regression analysis regarding SNP at position 128 showed that the 182CG heterozygotic genotype conferred risk of B-CLL (odds ratio, OR=3.8, 95% confidence interval, CI=2.4–6.1) that was further increased in alternative allele homozygotes (OR=7.3, 95%CI=2.8–19.1), p<0.0001. Similarly, compared to the individuals with wild-type 418CC genotype, carriers of heterozygotic 418CT genotype and alternative allele 418TT homozygotes were at higher risk of B-CLL (OR=7.3 and OR=240, respectively, p=0.032). Interestingly, we did not observe any differences in CD38 gene or protein expression in B-CLL or normal lymphocytes related to genotypes at position 128 or 418, however, this could be due to low numbers of rare genotypes found among patients included to the expression tests. Finally, the survival analysis showed that SNP at position 128 of CD38 gene influenced overall survival (OS) of B-CLL patients, with the best prognosis observed in carriers of 128CG genotype (p=0.05). In conclusion, our data indicate that inherited SNPs located in the CD38 gene are associated with genetic susceptibility to B-CLL in Caucasian patients, and may have some impact on prognosis of this malignancy.
Capture and Enrichment of CD34-Positive Haematopoietic Stem and Progenitor Cells from Blood Circulation Using P-Selectin in an Implantable Device.
Authors: Narasipura Srinivasa D. , Wojciechowski Joel C. , Charles Nichola , Mickelsen Deanne , Blair Martha L. , King Michael R.
Published: October 2019

Clinical infusion of haematopoietic stem and progenitor cells (HSPCs) is vital for restoration of haematopoietic function in many cancer patients. Previously we have demonstrated an ability to mimic physiologic cell trafficking in order to capture CD34-positive (CD34+) HSPCs using a monolayer of P-selectin in flow chambers. The aim of the current study was to determine if HSPCs could be captured directly from circulating blood in vivo. Vascular shunt prototypes, coated internally with P-selectin, were inserted into the femoral artery of rats. After one-hour blood perfusion, successful capture of mononuclear cells was achieved and analysis of these cells revealed a significant enrichment for CD34+ HSPCs. Blood flow through the cell capture device resulted in a wall shear stress of 4–6 dynes/cm2. Purity of captured CD34+ cells showed 7-fold enrichment (average purity = 28 ± 4%) over levels found in the control mobilized peripheral blood mononuclear cells (average purity = 3 ± 0.5%). Robust cell capture and HSPC enrichment were also observed in devices that were implanted in a closed-loop arterio-venous shunt conformation for two hours. Adherent cells were viable and were able to differentiate into burst forming units in culture. This study demonstrates an ability to mimic the physiologic arrest of HSPCs from blood in an implantable device and may represent a practical alternative for adult stem cell capture and enrichment.
Interleukin 11 Induced Capillary Leakage Syndrome and Deteriorated Hemorrhagic Cystitis in an Acute Myelogenous Leukemia Patient after Allo-HSCT.
Authors: Sun Xuemei , Yang Yonggong , Guan Chaoyang , Li Xiaohui
Published: October 2019

Interleukin 11 (IL11) has been approved for clinical utilization by Chinese Drug and Food Administration in the treatment of thrombopenia post chemo/radio therapy. It has been proved a safe cytokine in previous clinical trials. We however found a case who presented with a severe syndrome which was suspected in relation with IL 11. A 38- year-old male was undertaken a matched unrelated peripheral blood stem cell transplantation after an ablative BuCy conditioning for the treatment of acute myelogenous leukemia. He suffered from hemorrhagic cystitis(HC) on day 40 and was managed well with aggressive hydration and diuresis as well as administration of anti-virus regimen. After a resultant pancytopenia occurred, he was given IL11 for the amelioration of the thrombocytopenia in aid of controlling the bladder bleeding. His HC was in good control initially, but two days after the IL-11 administering, the patient manifested with generalized edema, hypotension with hemoconcentration and hypoalbuminemia without albuminuria. The diagnosis of capillary leakage syndrome (CLS) was under consideration. Interleukin 11 was ceased immediately and 6% hydroxyethly starch(HES) was added to improve the symptoms of CLS. In the following day the 24 hour- urinary volume of the patient reduced sharply accompanying by dysuresia. Ultrasonic examination displayed a clumping mass measured 50 mm×75mm ×63mm. Three days later, he was diagnosed as acute renal failure owing to combination factors of the renal and/or postrenal damage of the kidney. Meanwhile continuous renal replacement therapy (CRRT) was started and patient got well 5 days later with normal urinary volume. And symptoms from HC and CLS were improved dramatically. We concluded here that interleukin 11 could play the pivotal role in inducing a CLS and resultant acute renal failure. And it thus deteriorated HC in this patient. The patient’s renal function could recover well rapidly with the infusion of HES and CRRT. We suggested a comprehensive clinical study of Interleukin 11 be conducted to optimize its clinical usage for the sake of safe medication.
Tacrolimus and Short-Term Methotrexate (mini-MTX) for Graft Versus Host Disease (GVHD) Prophylaxis after Unrelated Single Unit Cord Blood Transplant (CBT) in Pediatric Patients.
Authors: Petropoulos Demetrios , Worth Laura L. , Koontz Susannah E. , Choroszy Mary S. , Chan Ka Wah , Shpall Elizabeth J. , Champlin Richard E. , Cooper Laurence J.
Published: October 2019

CBT is increasingly used in the management of patients with a variety of hematologic and neoplastic conditions. We investigated the use of Tacrolimus with MTX for the prevention of GVHD. Sixty consecutive pediatric patients (37 males, median age 6.8 years, range 0.4–20 y) underwent 63 single unit CBT. Sixty-two units were mismatched with the patients (1 Antigen mismatch [Ag MM]: 20; 2 Ag MM: 39; 3 Ag MM: 3). Five patients had non-malignant conditions (severe aplastic anemia [SAA] 2, Wiskott-Aldrich 2, Hunter 1). Thirty-one of the 55 patients with malignant diseases (ALL 30, AML 16, CML 2, NHL 2, MDS 2, JMML 2, neuroblastoma 1) had high-risk disease. The conditioning regimen was chemotherapy-only in 34 (Thiotepa, Busulfan[Bu], Cyclophosphamide[Cy] for 28 children younger than 5 years or with prior irradiation; Bu-Cy in the patient with Hunter and a patient with AML and trisomy 21; Fludarabine[Flu], Cy, Lymphoglobulin in one patient with SAA; Bu-Flu:3) transplants. Radiochemotherapy was implemented in 29 patients (Flu, Melphalan[Mel], TBI: 26; Etoposide, Mel, TBI:1; Etoposide, Mel, TBI:1; Flu, Cy, ATG, 200cGy TBI:1 SAA patient). The median pre-cryopreservation total nucleated cell dose was 4.3 x 107 / kg recipient weight (range 1.8–12 x 107/ kg). The combination of Tacrolimus and mini-MTX (5 mg/m2 on days 1, 3 and 6) was used in 60 transplants (Tacrolimus alone in 3). ATG or steroids were not routinely used.

Results: Forty-six of the 52 transplants evaluable for acute GVHD did not have severe GVHD (Grade 0:11 patients; I: 10 patients; II: 25 with 22 limited to skin only; III: 3; IV: 3 patients). Eight patients had primary graft failure (2 with persistent disease). There were 3 early deaths. Chronic GVHD was diagnosed in 8/42 evaluable patients. With a median follow-up of 1,757 days (range 512–3,731) 27 patients are alive without disease.

Conclusion: Tacrolimus with mini-MTX should be considered for the prevention of GVHD after mismatched unrelated cord blood transplants in pediatric patients with malignant or non-malignant diseases.
Nonmyeloablative Peripheral Blood Stem Cell Transplantation for Chronic Myeloid Leukemia in Chronic and Advanced Phases.
Authors: Ding Jia-Hua , Ma Yan , Chen Bao-An , Zhao Gang , Wang Jun , Sun Yun-yu , Cheng Jian
Published: October 2019

Objective: To study the effect of nonmyeloablative peripheral blood stem cell transplantation(PBSCT) for chronic myelogenous leukemia(CML) in chronic phase(CML-CP) and advanced phase(CML-AP).

Methods: Twenty-four CML patients, 16 in CML-CP and 4 in CML-AP underwent nonmyeloablative peripheral blood stem cell transplantation(NST). The conditioning regimen included fludarabine 30mg.m−2.d−1, intravenously(i.v.)×6d, busulphan 4mg.m−2.d−1×2d and CTX 350mg.m−2.d−1×2d combined with or without Ara-C. The donors were HLA-identical (n=20) and 5/6 antigen-matched (n=4).

Results: All the patients were successfully engrafted. The mean time needed for increase of the number of neutrophils to more than 0.5×109/L and platelet more than 20×109/L were 13 days and 11.5 days respectively. Among 12 patients, 9 showed complete donor chimerism(CDC) and 3 showed mixed chimerism(MC) at +day 30. At +day 180, 18 patients still alive showed CDC and remained alive after a median follow-up of 24 months (4∼48months). 3 cases died of severe acute GVHD(aGVHD), 1 case of chronic GVHD(cGVHD), 2 cases of interstitial pneumonia(IP) and 1 case of relapse.

Conclusion: Nonmyeloablative PBSCT is an effective method for CML patients in chronic phase and advanced phase.

Characteristics of 24 CML patients and their donors Patient Age Gender Diagnosis Donor HLA *: unrelated donor 1 40 male CML-AP brother HLA-identical 2 32 male CML-AP brother HLA-identical 3 40 male CML-CP brother HLA-identical 4 40 male CML-CP brother HLA-identical 5 42 male CML-AP brother HLA-identical 6 24 male CML-AP sister HLA-identical 7 55 male CML-CP sister HLA-identical 8 32 male CML-CP brother HLA-identical 9 36 male CML-CP brother HLA-identical 10 46 female CML-AP sister HLA-identical 11 52 female CML-CP brother HLA-identical 12 60 male CML-CP sister HLA-identical 13 45 female CML-CP brother HLA-identical 14 48 male CML-CP brother HLA-identical 15 33 male CML-CP URD* HLA-identical 16 35 female CML-CP URD HLA-identical 17 40 male CML-CP brother HLA-identical 18 34 female CML-AP sister HLA-identical twin 19 54 female CML-CP brother HLA-identical 20 33 male CML-AP URD Subtype mismatched 21 32 male CML-CP URD Subtype mismatched 22 42 male CML-CP URD mismatched 23 50 female CML-CP URD HLA-identical 24 46 male CML-CP brother HLA-identical
An In Vivo Evidence That the CD45negative Adult Marrow-Derived CXCR4+ SSEA-1+ OCT-4+ Very Small Embryonic-Like (VSEL) Stem Cells May Differentiate into CD45positive Long Term Repopulating Hematopoietic Stem Cells.
Authors: Wysoczynski Marcin , Kucia Magda , Zuba-Surma Ewa K. , Wan Wu , Ratajczak Mariusz Z. , Ratajczak Janina
Published: October 2019

Mounting evidence accumulates that bone marrow (BM) contains a population of pluripotent stem cells (PSC) that give rise to long-term repopulating hematopoietic stem cells (HSC). Recently we identified in murine BM a homogenous population of rare (∼0.01% of BMMNC) and very small (2–4 μm) Sca-1+ lin− CD45− cells that express by RQ-PCR and immunhistochemistry markers of PSC such as SSEA-1, Oct-4, Nanog and Rex-1 and highly express Rif-1 telomerase protein (Leukemia2006;20,857–869). Direct electronmicroscopical analysis revealed that these cells display several features typical for primary epiblast-derived embryonic stem cells such as i) a large nuclei surrounded by a narrow rim of cytoplasm, and ii) open-type chromatin (euchromatin). In co-cultures with C2C12 murine sarcoma supportive feeder-layer, these cells grow spheres that are composed of immature CXCR4+SSEA-1+Oct-4+ cells that have large nuclei containing euchromatin and, if plated into cultures promoting tissue differentiation, show pluripotency and expand into cells from all three germ-cell layers. Based on this, we called them very small embryonic like (VSEL) stem cells. However, VSEL isolated freshly from the BM do not posses immediate hematopoietic activity - they neither grow hematopoietic colonies nor radioprotect lethally irradiated recipients. Recently, however, we noticed that if plated over supportive OP9 cell line, these CD45− VSEL give rise to colonies of CD45+CD41+Gr-1−Ter119− cells where their phenotype resembles those of the earliest hematopoietic cells that are derived in vitro from established embryonic cell lines. This hematopoietic differentiation of VSEL was accompanied by upregulation of mRNA for several genes regulating hematopoiesis (e.g. PU-1, c-myb, LMO2, Ikaros). More importantly, the CD45+CD41−Gr-1−Ter119− cells expanded from VSEL isolated from GFP+ mice if transplanted into wild-type animals protected them from lethal irradiation and differentiated in vivo into all major hematopoietic lineages (e.g., Gr-1+, B220+ and CD3+ cells). This hematopoietic activity was maintained after transplantation into secondary recipients. Based on this, we postulate that i) VSEL are PSC that give rise to HSC and ii) that CD45+ cells may derive from a CD45− population. Thus we propose that VSEL are a population of BM-residing PSC that may give rise to long-term engrafting hematopoietic stem cells.
Hematopoietic Stem Cell Transplantation after Reduced Intensity Conditioning in Acute Myeloid Leukemia Patients Older Than 40 Years.
Authors: Huisman Cynthia , Meijer Ellen , Petersen Eefke J. , Lokhorst Henk M. , Verdonck Leo F.
Published: October 2019

Reduced intensity conditioning (RIC) protocols are increasingly used for allogeneic hematopoietic stem cell transplantation (HSCT) in elderly patients. We retrospectively analyzed the outcome of RIC HSCT in a homogeneous group of acute myeloid leukemia (AML) patients over the age of 40. Forty-three AML or high-risk myelodysplastic syndrome patients were treated with a fludarabine and low dose total body irradiation (TBI) based regimen, followed by a full peripheral stem cell graft. Antithymocyte globulin was given to matched unrelated recipients (34%) before infusion of fludarabine. Graft-versus-host disease (GVHD) prophylaxis consisted of cyclosporin and mycophenolate mofetil. All but 2 AML patients were in complete remission at the time of transplantation. Seventy-six percent of patients had a poor risk profile. Hematologic recovery was fast and primary graft failure occurred in one patient. Two patients with active disease at the time of HSCT experienced ongoing relapse. Infections were diagnosed in 9 patients (21%) and 6 patients (14%) were treated for CMV reactivation. Sixty percent of patients developed acute GVHD, which was grade 2 in 40% and grade 3 in 12%. Chronic GVHD occurred in 33% of patients. The incidence and severity of both acute and chronic GVHD was similar in patients with related and unrelated donors (P = 0.84 and 0.74, respectively). Treatment-related mortality (TRM) was low (9%), total nonrelapse mortality was 19%. After a median follow-up of 571 days, 16 patients (37%) experienced relapse. One-year progression-free and overall survival were 61% and 67%, respectively. Median disease-free survival has not been reached yet (> 58 months) and median overall survival was 31 months (Fig 1 and 2, respectively). Poor risk AML was significantly associated with disease-free survival in multivariate analysis (P = 0.02). Age > 60 years, gender, donor type, timing of RIC HSCT (upfront or after relapse) and acute GVHD were not identified as prognostic factors. In conclusion, fludarabine plus low-dose TBI-based RIC HSCT is effective in AML patients over the age of 40 without active disease at the time of transplant and is associated with low TRM.

Figure Figure Figure Figure
Brazilian Experience Using High Dose Sequential (HDS) Followed by Autologous Hematopoietic Stem Cell Transplantation (AHSCT) for Relapse/Refractory Aggressive Non-Hodgkin Lymphoma (NHL).
Authors: Duarte Bruno K.L. , Valente I.S. , Vigorito Afonso C. , Aranha Francisco J.P. , Oliveria Gislaine B. , Miranda Eliana C.M. , Lorand-Metze Irene , Pagnano Katia B.B. , De Souza Carmino A.
Published: October 2019

The purpose of this study was to evoluate the use of high dose cyclophosphamide HD (CY) 7 or 4g/m2 and HD etoposide (VP-16) 2g/m2 followed by AHSCT in patients affected by aggressive NHL. From January 1998 to November 2006, 106 patients, 66 males, 40 females, median age 47 (8–66) years underwent into this regimen. The diagnosis was 83 (78.3%) DLBCL, 13 (12.3%) with T-cell Lymphoma and 10 (9.4%) Mantle Cell. At diagnosis, 88 (83%) were stage III or IV, 34 (32.1%) presented bone marrow involvement, 65 (61.3%) bulky disease, 67 (63.2%) B symptoms and 45 (42.5%) were high or high intermediate risk according to IPI. Prior to HDCY patients had been submitted to a mean of 2 (1–3) therapy lines and 6 (5.7%) were in complete remission (CR), 38 (35.8%) partial remission (PR), 62 (58.5%) disease progression (PD) or relapse. Concerning CY dose 42 (39.6%) patients received 4 g/m2 and 64 (60.4%) 7 g/m2, respectively. After HDS, 13 (31%) patients who had used 4 g/m2 and 30 (47%) used 7 g/m2 were in CR (P= 0.03). After the HDCY 43 (40.6%) were in CR, 33 (31.1%) PR, 21 (19.8%) remained in PD and 9 (8.5%) died. After a median follow-up of 4.1 (1.5–57) months, 80 patients (75.5%) were submitted to AHSCT and their present status is 44 alive [33 CR, 4 PR, 7 PD]; 36 dead [4 CR, 1 PR, 2 relapse, 17 PD and 12 had death related to procedure]. Their overall survival was 45% in 8 years. Currently we have 50/106 (47%) alive patients, 35 CR, 6 PR and 9 PD. Overall Survival (OS) was 37%, Disease Free Survival (DFS) 49%, Progression Free Survival (PFS) 42%, Event Free Survival (EFS) 28%. OS by diagnosis was 42% DLBCL, 40% T-cell in 8 years whereas 20% Mantle Cell in 6 years (P=NS). OS by B symptoms patients was 22% versus 58% (P= 0.002) and EFS was 23% versus 37% (P= 0.03). Patients who were in PD prior to the HDCY compared to their status after that had a significant improvement (P< 0.001), their OS was 38% to CR-PR group (38) versus 0% PD group (24). In general, mortality was 56 (53%), their cause was 23 (41.1%) PD, 7 (12.5%) related to HDCY, 12 (21.4%) related to AHSCT, 13 (23.2%) infections and 1 (1.8%) GVHD a RIC transplant. Besides that 1 (0.9%) patient developed MDS and is alive. B symptoms at diagnosis have appeared as a predictor factor for survival. Our study suggests HDS is an efficient treatment to improve status and to reduce tumor burden. Although it presented high toxicity related mortality, we consider the treatment feasible, especially considering the patients’ poor prognosis.
Immune Mobilization with Direct In-Vivo Effector Response: Clinical Trial with Favorable Implications for Post-Transplant Outcome.
Authors: Hill John M. , Wu Jia-Yan , Webber Susan M. , Root Lynn D. , Kimtis Elizabeth A. , Williams Idalina C. , Ernstoff Marc S. , Szczepiorkowski Zbigniew M. , Sentman Charles L. , Meehan Kenneth R.
Published: October 2019

Autologous stem cell transplant is an effective modality for many patients with lymphoproliferative disorders. Still, the majority of patients with myeloma and many with lymphoma relapse after transplant. Innovative post-transplant immunotherapies are needed. We initiated a phase I immune mobilization trial utilizing dose escalation of IL-2, in combination with GM-CSF and G-CSF, in an attempt to mobilize autologous cytotoxic effector cells, along with peripheral CD34+ hematopoietic progenitor cells. IL-2 began on day 0 of mobilization and continued as a daily SQ injection for 11 days. On day 7 of mobilization, GM-CSF (7.5 mcg/kg/d) and G-CSF (5 mcg/kg/d) were initiated for 5 days (day 7–11). On day 11, leukapheresis was performed. Patients then received melphalan (200 mg/m2) followed by reinfusion of cryopreserved autologous peripheral hematopoietic progenitor cells. Phenotypic and functional analyses were performed using peripheral blood mononuclear cells (PBMNC) collected during mobilization on days 0, 7 and 11. Eleven of 12 patients treated to date completed the regimen (myeloma, n=11; NHL, n=1). One patient (NHL) was removed due to progressive disease. The first two dose levels of IL-2 have been well tolerated (dose level 1: 6x105 IU/m2/d; n=6 pts; dose level 2: 1x106 IU/m2/d; n=6 pts) with no ≥grade 3 toxicities noted. Dose escalation of IL-2 continues since the MTD has not been reached. Phenotypic analyses of PBMNC demonstrate an increase in CD3+CD8+ T cells from 17.5% (day 0 baseline) to 22.7% (day 11; p = 0.01). CD56+ NK cells increased from 18.9% (day 0) to 36.0% (day 11; p = 0.002), and CD8+CD56+ NKT cells increased from 8.2% (day 0) to 18.0% (day 11; p = 0.01). Cytotoxicity directed against a human myeloma cell line using peripheral blood lymphocytes was 8.6% at baseline and increased to 43% on day 11 of mobilization (p=0.03). All patients successfully mobilized and received an autologous transplant without delay in engraftment (ANC recovery: day 13 median; range 10–14 days; platelet recovery: day 12 median; range of 0–13 days). These results are encouraging and demonstrate effective mobilization with minimal toxicity and marked in vivo immunomodulatory effects. In addition, the enhanced cytotoxic effector cell number and killing of myeloma cells is quite promising, with additional follow up ongoing to determine the potential impact on survival.
Effects of Cyclosporine A on Lentiviral Transduction of Mouse Hematopoietic Stem Cells and Transplantation Using Nonmyeloablative Conditioning.
Authors: Sutherland Nadia D. , Spencer H. Trent
Published: October 2019

Achieving high level transduction of murine hematopoietic stem cells (HSCs) using lentiviral vectors has been a challenge for many laboratories. We investigated the efficiency of lentiviral transduction of murine stem cell antigen-1+ (sca-1+) cells with and without cyclosporine A (CSA), which has previously been shown to increase the transduction efficiency of other types of murine cells. Sca-1+ cells were isolated from C57BL/6 mice and transduced with lentiviral vectors encoding green fluorescent protein (GFP) at various multiplicity of infections (MOI) and with various concentrations of CSA. Twenty-four hours after a single transduction, 1.5 x 104 or 4.5 x 104 cells were plated in methylcellulose containing the appropriate cytokines for progenitor cell growth, and colonies were counted on days 8–12. In the absence of CSA only 4± 2% of progenitor colonies were GFP+. However, when CSA (10 μM) was added within 3 hours of sca-1 cell isolation, transduction efficiency increased to 44 ± 6%. Although the transduction efficiency increased 10-fold, the number of progenitor colonies significantly decreased when CSA was added (up to 90% decrease). Lower concentrations of CSA (e.g. 1 μM) were less toxic to sca-1+ cells but resulted in inconsistent transduction efficiencies. We next determined the effects of CSA when applied at various times after sca-1 cell isolation. We found that the number of sca-1+ cells decreased within the first two days of culture but then begin to increase on day 3, and by day 7 there is a 7-fold increase compared to the number of cells originally isolated. Cells cultured with virus alone had an average increase of 3.5-fold on day 7, but only 3% of cells cultured in CSA survived to day 7. Cells cultured with both virus and CSA had no viability on day 7. However, by delaying the addition of virus and CSA until day 3, a 1.4-fold increase in sca-1+ cells was observed by day 7, which was achieved without affecting the efficiency of transduction. Sca-1+ cells were then transduced with the lentiviral vector in the presence of CSA and transplanted into transgenic sickle mice using a nonmyeloablative conditioning regimen that consisted of busulfan (25 mg/kg) administered on day -1 and costimulation blockade with CTLA-4Ig and anti-CD40 ligand administered on days 0, 2, 4, and 7. We were able to achieve donor engraftment levels of 98% with a 40% engraftment of gene-modified cells. These results show that using CSA in lentiviral transductions of murine HSCs can be an effective method for increasing overall transduction efficiency, and may aid in the use of lentiviral vectors in animal studies.
Drug Utilization Patterns and Cost Considerations for Erythropoiesis Stimulating Agents in Cancer Chemotherapy Patients in a Managed Care Setting.
Authors: Mandel Alyson , Weininger Richard , McKenzie Scott R. , Bookhart Brahim K. , Piech Catherine T.
Published: October 2019

Objective and Purpose: To understand current utilization patterns, this study examined real-world dosing and drug costs for erythropoiesis-stimulating agents (ESAs), epoetin alfa (EPO) and darbepoetin alfa (DARB) in cancer patients receiving chemotherapy.

Methods: An analysis of ESA utilization in chemotherapy patients using adjudicated medical claims between 2004 and 2006 from seven health plans was conducted. Patients with ≥1 ESA claim, receiving concurrent chemotherapy identified through alphanumeric HCPCS codes, and newly initiated on EPO or DARB (no ESA administration within 90 days prior to initiation), and receiving chemotherapy were included. Those patients who received both EPO and DARB or who had myelodysplastic syndromes or end-stage renal disease were excluded. Treatment duration was defined as time from first to last ESA administration. Mean cumulative ESA dose was used to calculate drug cost (based on 1/2007 wholesale acquisition cost: EPO $12.52/1000 Units; DARB: $4.576/mcg) and dose ratio (Units EPO: mcg DARB).

Results: 1,446 EPO and 2,729 DARB patients were included. Mean treatment duration was longer in the DARB-treated group by three days (EPO:49 days; DARB:52 days; p= 0.037). The EPO-treated group reported 5.8 administrations with a mean administered dose of 42,603 Units (median 40,000, interquartile range 40,000–45,714) corresponding to a mean cumulative dose of 247,097 Units. The DARB-treated group reported 5.0 administrations with mean administered dose of 262 mcg (median 250, interquartile range 200–300) corresponding to a mean cumulative dose of 1,310 mcg. Drug costs were $2,901 lower in the EPO group compared to the DARB group (EPO $3,094; DARB $5,995) and utilization reflected a dose ratio of 189:1 (Units EPO: mcg DARB).

Conclusions: This observational study of over 4,100 cancer patients receiving chemotherapy found 48% lower drug cost in the EPO group compared to the DARB group. These findings provide greater understanding of current real-world ESA utilization and are consistent with other studies in the cancer chemotherapy population.
Treatment of Leukemic Meningeosis by CD 14 Depleted Adoptive Intrathecal Cell Therapy.
Authors: Thiel Eckhard , Blau Igor W. , Neumann Martin , Tietze-Buerger Carola , Gerbitz Armin , Uharek Lutz
Published: October 2019

Background: Leukemic relapse within the central nervous system (CNS) is generally difficult to treat and results in poor outcome. Limiting factors for effective treatment are mainly extended neurotoxicity and availability of only few agents that will effectively act across the blood brain barrier (BBB). Although graft versus leukemia effects after allogeneic stem cell transplantation are well documented, its application is not recommended in patients with CNS disease. Since the CNS represents an immunologically privileged organ, conventional donor lymphocyte infusion (DLI) has not proven to be effective in CNS relapse situation after stem cell transplantation. Here we present results from three patients suffering from isolated CNS relapse of CML or AML after allogeneic stem cell transplantation receiving for the first time intrathecal cell therapy using CD14 depleted peripheral blood mononuclear cells from their allogeneic donor. Besides leukemic blast cell counts of the CSF molecular genetic analyses for chimerism as well as for translocations were applied for monitoring in addition to MRI and to neuroclinical symptoms.

Results: Up to eight consecutive CD14 depleted intrathecal infusions were applied in escalating doses starting at 1x106 CD3+ cells. In all cases we did not observe any immediate or delayed side effects post application. In the case of the CML patient we observed disappearance of the leukemic blasts and of bcr-abl translocations and reversal to full donor chimerism of the CSF. In one AML patient we observed a transient disappearance of leukemic blasts, followed by a delayed increase. No clear and sustained response was recorded in the other AML patient. All patients are still under observation (9–14 months) and remained full chimeras in peripheral blood and bone marrow; however, the AML patients meanwhile developed chloroma lesions.

Conclusion: Intrathecal DLI for CNS relapse of leukemia after allogeneic stem cell transplantation is a new still experimental approach. The first applications indicate no side effects e.g. cerebral or meningeal GVHD and a possible efficacy in CNS relapse of CML. This new approach merits further investigation in the setting of a clinical trial.
Genetic Polymorphism of Cytochrome P450 1B1 (C432G) Is Associated with An Increased Treatment-Related Mortality and Lower Overall Survival in Patients Undergoing Allogeneic Hematopoietic Stem Cell Transplantation.
Authors: Koldehoff Michael , Elmaagacli Ahmet H. , Beelen Dietrich W.
Published: October 2019

Background. The human cytochrome P450 1B1 (CYP 1B1) is a key enzyme involved in the production of reactive metabolites and in the activation of environmental carcinogens. Several polymorphisms were identified in CYP 1B1 gene; four of them are single nucleotide polymorphisms and give rise to amino acidic substitutions. The CYP 1B1 codon 432 polymorphism leads to a three-fold higher 4-hydroxylase activity for the variant CYP 1B1 isozymes than the wild types. Never before the influence of genetic polymorphisms of CYP 1B1 (C432G) on patients who underwent allogeneic hematopoietic stem cell transplantation (HSCT), was evaluated.

Methods. Here we genotyped in a retrospective study 384 recipients (R) (and their donors (D)) for CYP 1B1 (C432G) expression that underwent allogeneic HSCT for various diseases and analyzed their outcome. Genotyping of CYP 1B1 (C432G) was performed by real-time PCR.

Results. 170 R (44.3%) were genotyped as homozygous wild-type gene C/C, 157 R (40.9%) were genotyped as heterozygous genotype C/G and 57 R (14.8%) were genotype as homozygous gene mutation G/G. From the 167 D (43.5%) were C/C, 164 D (42.7%) were C/G, and 53 D (13.8%) had a homozygous gene mutation G/G. A homozygous CYP 1B1 gene mutation G/G was found on 18 R/D side (4.7%). Calculated genotype frequencies did not differ from that reported earlier by other studies for Caucasians. Five-year estimate for treatment-related mortality (TRM) and overall survival (OS) were statistically different in genotype C/G- and G/G- R with 33 ± 4%, and 49 ± 4% compared to homozygous wild-type gene C/C- R (18 ± 3%, and 59 ± 4%, respectively, [p<0.03]), whereas the five-year estimate for relapse rate (RR) was not different between the groups. No differences for five-year estimates for TRM, RR, or OS were seen in R with either genotype C/C-, C/G- or G/G- D. No statistic differences were found in the incidence of acute GVHD grade 2–4 on R- or on D- side with variant CYP 1B1 (C432G) polymorphisms. Surprisingly, the five-year estimate for TRM, RR, and OS were statistically different in homozygous gene mutation G/G on R/D site with 57 ± 2%, 81 ± 2%, and 16 ± 1% compared to all other CYP 1B1 genotypes with 28 ± 3%, 26 ± 2%, and 55 ± 3%, respectively [TRM, p<0.01; RR and OS, p<0.001]). Multivariate analysis confirmed that CYP 1B1 (C432G) homozygous gene mutation G/G on R/D site had an increased risk for TRM and RR (p<0.02), whereas the mutation G/G on R/D site revealed a worse OS (p<0.01).

Conclusions. These results suggest that recipients with genetic polymorphism of CYP 1B1 do have an increased TRM, RR and lower OS after transplantation. Genotyping for CYP 1B1 (C432G) might help to identify patients with higher risk for allogeneic transplantation.