bluuebunny/crossref_metadata_2025 huggingface dataset:
Successful Treatment of Cutaneous Chronic Lymphocytic Leukemia with a Topical Toll Receptor 7/8 Antagonist (Imiquimod).
Authors: Hicks Lisa , Mena J. , Spaner D. E.
Published: April 2020

Background: B cells express toll-like-receptor-7 (TLR7), a powerful modulator of inate immunity. We hypothesized that the immunogenicity of indolent B cell lymphomas and leukemias could be increased through activation of the TLR7 pathway. This might lead to increased immune clearance of malignant cells. Imiquimod 5% (an imidazoquinolone) has recently been identified as a TLR 7/8 agonist. It is available as a topical formulation and has demonstrated activity against condylomata as well as skin carcinomas. A small number of case reports also suggest activity against cutaneous T cell lymphoma.

Case: A 71 year old Caucasian man presented with Rai Stage 0 CLL. For approximately 8 years prior to presentation, he suffered recurrent, erythematous, nodular lesions on his hands, arms and torso. The lesions were removed with liquid nitrogen but recurred. At the time of diagnosis with CLL, several lesions were present. Biopsy demonstrated a diffuse infiltrate of small round lymphocytes without epidermotropism. CD20 staining was positive. Molecular analysis confirmed a monoclonal B cell population consistent with B cell lymphoma. The patient was otherwise asymptomatic and did not warrant conventional therapy for CLL. Based on the safety and efficacy of imidazoquinoline in other skin cancers, he was offered and consented to a trial of this therapy. The drug was applied to the affected area 3x per week as recommended for treatment of genital warts. After 8 weeks, an area of hypopigmentation had formed, however the size of the lesion had not decreased. The frequency of application was increased to 1x per day. Over a 6 week period the lesion gradually disappeared. Six months after stopping the drug, the lesion has not recurred. No side effects were noted. Untreated lymphomatous lesions and peripheral blood lymphocyte counts did not change over the course of treatment.

In vitro Data: We hypothesized that Imiquimod might increase the immunogenicity of CLL cells by increasing their surface expression of costimulatory molecules. To investigate this hypothesis, the above patient’s CLL cells were isolated (with his consent) directly through negative selection and then incubated for 48h with an active soluble imidazoquinalone or with a control. Expression of co-stimulatory molecules was determined by flow cytometry pre and post-incubation. CD80, 83, 86 and 54 surface expression were found to be increased significantly by TLR-7 agonists in vitro.

Conclusion: We report the first case of Imiquimod activity against a cutaneous B cell lymphoma. We propose that the drug increases co-stimulatory molecule expression by malignant B cells, thereby facilitating cytotoxic T cell activation and killing of CLL cells. We believe that further study of TLR7 agonists is warranted in B cell malignancies.
What Is Important Factor of Bone Disease in Multiple Myeloma?.
Authors: Munemasa Shoso , Sakai Akira , Okikawa Yoshiko , Kuroda Yoshiaki , Katayama Yuta , Asaoku Hideki , Kimura Akiro
Published: April 2020

New International Prognostic Index (IPI) staging system of multiple myeloma (MM) is a combination of the level of serum β2-microglobulin and serum albumin. Particularly, good survival (median survival >5 years) is associated with absence of chromosome 13q deletion. Recently, correlations between molecular subtypes and prognosis have been identified as a good prognosis with t(11;14) and a poor prognosis with t(4;14) and t(14;16) besides chromosome 13 abnormalities. We have reported that some MM cases with cyclin D1 overexpression detected by competitive RT-PCR were not caused by t(11;14)(q13;q32) or extra copies of 11q13 (In J Oncol, in press). A recent report revealed that subtypes of MM cases with the translocation of cyclin D showed a close correlation with bone disease and high level of DKK1. We also have been studing about the correlation between bone disease and bone morphogenetic protein (BMP) 2, or connective tissue growth factor (CTGF) that is supposed to inhibit the VEGF binding to its receptor or modulate cell signaling by BMP. First, we analyzed IPI staging in 91 MM cases, and then analyzed the relation between IPI staging and existence of cyclin D1 overexpression, or t(11;14)(q13;q32) and extra copies of 11q13. Competitive RT-PCR was performed in 77 cases, and cyclin D1 overexpression was detected in 40/77 (52%). Deletion of chromosome 13q was detected in 32/87 (37%), and t(11;14)(q13;q32) or extra copies of 11q13 was detected in 11/50 (22%) and 7/50 (14%), respectively. There were no significant differences of those factors among IPI staging. And we analyzed the scale of bone lesion by bone x-ray in 81 cases. We could not detect the relation between bone disease and cyclin D1 overexpression or translocation of 11q13. Furthermore, we analyzed the expression of BMP2 and CTGF by quantitative real time-PCR in purified myeloma cells or in bone marrow mononuclear cells (BMMNC) reduced myeloma cells less than 5%. We have gotten results that MM cases have a tendency to show higher CTGF expression in BMMNC compared with that of normal BM, but there was no significant difference of BMP2 expression in BMMNC between them. And there was no correlation between cyclin D1 overexpression and BMP2 or CTGF expression. So far a cause of bone lesions in MM is supposed to be the activity of osteoclast, however, our preliminary examination by TRAP staining revealed that osteoclast differentiation from BMMNC in MM cases by adding M-CSF (25 ng/ml) and RANKL (50 ng/ml) decreased compared with that in normal BM, and osteoblast diffentiation also decreased in MM by cytochemical staining for alkaline phosphatase (AP). We guess that both osteoclast and osteobalst differentiation are suppressed in MM and CTGF is a candidate for the suppressor of osteoblast differentiation. We will be able to show the result of AP activity of osteoblast and the effect of recombinant CTGF on osteoblast in meeting.
An Edible, High Molecular Weight Glycoprotein Fraction from Ganoderma Lucidum Inhibits the Akt Pathway in Resistant Myeloma Cell Lines Decreasing Mcl-1 Expression, Up-Regulating FKHR and Inducing Apoptosis.
Authors: Sultana Nazneen , Verda Larissa , Burt Richard K. , Traynor Ann E.
Published: April 2020

We observed apparent synergy between dexamethsone and a nutritional supplement taken by a patient with poor prognosis plasma cell leukima. The rapidity of the clinical remission induction and the duration of remission, which was sustained without consolidation, suggested possible inhibition of the Akt pathway. A review of the supplements ingested indicated two mycelial derivatives, one of which was Ganoderma Lucidum, (Ling-Zhi, Reishi) a traditional Chinese medicine used to prevent kidney and liver disease and to promote anti-tumor immunity. We obtained a known quantity of certified shell broken spore of G Lucidum from China. Dissolved in 70 degree C water and then diluted as a 2.5% v/v solution in RPMI1640, it induced apoptosis of 80% of OPM6 cells by 72 hours, which was only marginally improved by the addition of1uM dexamethasone. It induced 70% cell death in MM.R cells by 72 hours. This was associated with a 90% reduction in measurable Mcl-1 by Western blot after two hours of incubation. In additon phosphorylation of FKHR was reduced 70% at 2 hours and was undetectable after 72 hours. Phosphorylation of Akt at threonine 308 was reduced 75%. To further purify this activity we separated out molecular weight fractions by Centricon centrifugation. We were able to 10 fold concentrate the active material in the >100,000 MW fraction by centrifugation. Used in the same relative dilution of 2.5% v/v, this concentrate killed between 90% and 96% of glucocorticoid resistant cells at 72 houyrs. Of interest the concentrate induced a lesser degree of apoptosis in corticosteroid sensitive cells, but enhanced the activity of dexamethasone. Spectrophotometry followed by oxidation with sodium meta-periodate and measurement absorption at 550 nm suggested a glycoprotein enrichment in this concentration. Further isolation of the active component of this fraction is underway in order to determine if it is the Lin-Zhi 8 protein sequenced in 1991 or a distinct glycoprotein from the Ganoderma Lucidum. It appears to be an orally tolerable inhibitor of the Akt pathway. It also shows activty in rheumatoid arthritis synovium RAS cells and may have usefulness in T cell supression and graft tolerance.Pre-clinical testing in a SCID mouse model has been begun.
Frascati , a Mitochondrial Solute Transporter, and Its Role in Vertebrate Erythropoiesis.
Authors: Paw Barry H. , Shaw George C. , Cope John J. , Corson Kenneth , Hersey Candace , Wingert Rebecca , Ackermann Gabriele E. , Traver David , Trede Nikolaus S. , Barut Bruce A. , Zon Leonard I.
Published: April 2020

Acquired and congenital defects in iron metabolism from either deficiency or excess are one of the most common human diseases. Here we present the characterization of the zebrafish frascati mutation, which results in a profound hypochromic anemia and a developmental arrest at the pro-erythroblast stage. Using a positional cloning strategy, we have identified the gene disrupted frascati in mutants as a novel member of the mitochondrial solute transporter family. Members of this family of solute carriers have related tripartite sequence and structure. They function in transporting various metabolites and substrates across the inner mitochondrial membrane. We have verified the identity of the gene in zebrafish by the following criteria: (a) tissue-restricted expression in erythroid progenitors, (b) identification of missense mutations from the frascati five alleles, (c) rescue of anemia by over-expression frascati of cRNA in mutant embryos, and (d) mimicry of anemia using inactivating antisense morpholinos in wildtype embryos. We have also identified the functional ortholog in the mouse which has an analogous tissue and developmental expression pattern. The frascati ortholog in the mouse is highly expressed in fetal liver and adult bone marrow and spleen. The murine frascati transcript and protein are induced during terminal erythroid differentiation in MEL cells treated with either DMSO or HMBA. The over-expression of the mouse frascati cRNA in zebrafish frascati mutant embryos rescued their anemia with equal efficacy as the zebrafish clone. Given the identity of the gene and the requirement for iron in heme biosynthesis in the mitochondria of the developing erythron, we injected exogenous iron-dextran into frascati embryos. The embryos injected with iron-dextran were allowed to develop to 3 days post-fertilization, then stained for hemoglobinized cells with o-dianisidine and genotyped. Using this assay, the anemia caused by frascati the mutation could be partially rescued with exogenous iron supplementation. We therefore propose that the frascati gene functions as the essential transporter for iron importation into the mitochondria for heme biosynthesis and subsequent hemoglobin production in developing erythroid progenitors. Insight into the function of frascati the gene will be directly relevant to our understanding of human disorders of iron deficiency anemia and iron-overload sideroblastic anemia.
Osteonecrosis of the Jaw Associated with Pamidronate Therapy.
Authors: Zarychanski Ryan , Elphee Erin , Embil John , Walton Paul , Johnston James B.
Published: April 2020

BACKGROUND: Bisphosphonates, as a class of medication, fulfill an essential role serving to modify the natural history of bone disease associated with multiple myeloma and other neoplasms that infiltrate bone. They are also commonly used for the prevention and treatment of osteoperosis. Bisphosphonates are generally well tolerated and associated with minimal adverse effects; however, there exists a growing concern that bisphosphonate use may be associated with osteonecrosis of the jaw.

CASE PRESENTATION: We report the occurrence of osteonecrosis of the jaw associated with pamidronate therapy in 4 patients undergoing treatment for multiple myeloma. At the onset of jaw osteonecrosis, pamidronate therapy was the single medication common to all 4 patients. The duration of therapy varied from 18 to 65 months before osteonecrosis was observed. All cases involved the posterior mandible and have been refractory to a variety of medical therapies including surgical debridement and systemic antibiotics. Biopsies available from two patients revealed inflammation consistent with osteomyelitis. In one biopsy Actinomyces spp. were recovered from culture, but treatment with an extended course of clindamycin conferred no clinical benefit. The persistence of exposed bone remains a significant source of morbidity and pain for each patient. Discontinuation of pamidronate therapy has not helped to reverse the presence of osteonecrosis and surgical manipulation of the involved site appears to worsen the underlying bone pathology.

CONCLUSIONS: Osteonecrosis of the jaw is an important adverse outcome associated with bisphosphonate therapy. The unremitting nature of this clinical development, and the long lasting morbidity associated with it suggests that patients should be counseled regarding the possible occurrence of osteonecrosis prior to initiating therapy with pamidronate.
GvHD and Engraftment after Haploidentical Stem Cell Transplantation. A Comparison of 3 Immunomagnetic Selection Procedures.
Authors: Lang Peter , Schumm Michael , Greil Johann , Bader Peter , Handgretinger Rupert , Klingebiel Thomas , Schlegel Paul Gerhard , Niethammer Dietrich
Published: April 2020

Profound depletion of T and B cells is a fundamental prerequisite for haploidentical transplantation. Here, we compare hematopoietic engraftment and occurrence of GvHD after two different positive selection procedures (with anti-CD34 coated or anti-CD133 coated beads) and after a direct depletion procedure with CD3/CD19 coated microbeads in children with leukemias (ALL, AML, CML) and lymphomas. Median purity of stem cells was comparable after CD34+ selection and CD133+ selection, whereas stem cells were only slightly enriched after CD3+/CD19+ depletion (97.5%, 93.4% and 1.02%). Indirect depletion of T cells was better after CD34+ selection than after CD133+ selection (4.1log vs. 3.75log, p<0.001). Direct T cell depletion with anti-CD3 coated microbeads produced results comparable to CD34+ selection. Those grafts comprised also remarkable amounts of effector cells such as NK cells (median number: 40 x106/kg), dendritic cells and monocytes (median number: 120 x106/kg). Recovery of stem cells was similar in all three methods (77.3%, 80.6%, and 70% respectively).

Primary engraftment occurred in 85% (44/52) of patients with CD34+ selected stem cells and in 78% (11/14) of patients with CD133+ selected cells. After reconditioning, sustained engraftment was achieved in 98% and 100%. All patients (10/10) with CD3+/CD19+ depleted grafts had primary engraftment. Recovery of platelets was rapid especially in patients with CD133+ selected grafts (median time to reach independence from substitution = 14.5 days). GvHD grade II occurred in 6%, 7% and 25%, respectively. Only one patient had severe GvHD grade III-IV (CD34+).Conclusions: CD34+ selection and CD133+ selection resulted in similar engraftment and GvHD rates. CD3+/CD19+ depletion resulted in better engraftment but produced slightly more GvHD. Thus, the methods may be individually used according to the patient’s requirements.
Fludarabine Phosphate-Based Nonmyeloablative Stem Cell Transplantation in the Elderly Patients: A BHS Study.
Authors: Bron Dominique , Zachée Pierre , Maertens Johan , Theunissen Koen , Ferrant Augustin , Martiat Philippe , Doyen Chantal , Noens Luc , Schroyens Wilfried , Sellesslag Dominique , Demuynck Hilde , Schots Rick , De Meuter Anne , Boogaerts Marc
Published: April 2020

Background: Fludarabine phosphate (F)-based nonmyeloablative conditioning regimens are associated with reduced transplant-related toxicity and allow transplantation in patients (pts) above 55 y.o.

Aims: We prospectively studied the role of ATG to control the development of graft-versus-host-disease (GVHD), accelerate donor engraftment and maintain GVT effects in pts not eligible for classical allogeneic transplant. The subgroup of pts with 60 yo or more was analysed and compared with the younger population.

Population: A total of 113 pts were enrolled in the phase II study. Pts with lymphoid malignancies were conditioned with F (30 mg/m²/day for 4 days) plus cyclophosphamide (1g/m²/day for 3 days) or cytarabine (Ara-C, 2g/m²/day) for myeloid malignancies. All pts received cyclosporine (3–5 mg/kg IV, daily) and ATG (10 mg/kg/day for 4 days - [ATG-4, n= 36] or 2 days - [ATG-2, n=67] ). Chimerism analyses were performed on d30, 45, 60 and 90.

Results: 29 pts had 60 y.o. or more.Diagnoses were as follows: 9 MM, 5 NHL, 3 CLL, 5 AML, 4 MDS, 2 CML, 1 WM. The median age was 63 (range 60–74) years. The median follow-up was 24 months. Prior to transplantation, 35% of pts were in complete remission. 65% had poor prognostic disease(PR, RR or PD). Engraftment rate was 100% of the evaluable pts. Treatment-related mortality was 48%,much higher than in the younger population (15%) and due primarily to infectious complications. Grade II-IV acute GVHD (aGVHD) at d90 were similar in both populations (35%). Chronic GVHD rates were similar in both groups (40%). At d90, 23 pts were evaluable for T-cell chimerism analysis. Full (>90%) donor chimerism was achieved in 20/23 pts. Overall survival was higher in patients with good prognostic diseases (60% vs. 48%). No pts developed veno-occlusive disease.

Conclusion: These results confirm the feasibility of F-based nonmyeloablative conditioning in elderly population but infectious complications remain a concern compared to younger population.
Less Treatment Related Mortality with Variable Intensity Conditioning Than Non-Myeloablative Conditioning in Unrelated and Related Adult Allogeneic Transplant Recipients.
Authors: Bigelow Carolyn L. , Elkins Stephanie , Hardy Cheryl L. , Files Joe Clark
Published: April 2020

For the past four years our adult allogeneic transplant program has employed two alternative approaches to standard recipient conditioning, the use of non-myeloablative “mini” conditioning and variable intensity conditioning. We now report a retrospective comparison of relapse, day +100 and one year survival, engraftment and grades I-II and III-IV acute GVHD in unrelated as well as related recipients in these two preparative regimen groups. Patients with a variety of malignancies were not randomized to receive either non-myeloablative (Group 1) or variable intensity (Group 2) conditioning. Twenty patients with a median age of 49 (range 27–64, Group 1) and 17 patients also with a median age of 49 (range 24–58, Group 2) received either marrow or peripheral blood stem cells, usually with a 6/6 match grade; one recipient in Group 2 received a cord blood transplant (4/6 match). Group 1 regimen consisted of fludarabine 30 mg/m2 x 3d and TBI 200 cGY. Group 2 regimen consisted of Campath 20 mg/d either 5 or 3 days, fludarabine x 5d and melphalan 140 mg/m2 x 1d. GVHD prophylaxis was the same in both groups (standard dose cyclosporine or tacrolimus and MMF.) All patients received an adequate CD34+ cell dose and none of the products was manipulated. Relapse rate was 37% in Group 1 and 53% in Group 2. Day+100 survival and one year survival were 55% and 20%, respectively, in Group 1 vs 69% and 33% in Group 2. Only one patient in Group 2 had acute GVHD, grades I-II; none had grades III-IV. However, in Group 1, 6 patients had grades I-II and 8 had grades III-IV (40%). Graft failure occurred in five patients in Group 1, while no patients in Group 2 experienced it. We conclude, first, that in our program the application of variable intensity conditioning has been quite successful in unrelated transplant recipients, as well as in related. Second, significant treatment related mortality in the form of graft failure and acute GVHD occurred less frequently in recipients who received this conditioning than in those receiving non-myeloablative conditioning. This regimen requires some further modification to enhance its tumoricidal properties; however, its treatment-related toxicity is minimal and allows us to offer this therapy to patients with co-morbid conditions and older age.
Valganciclovir for the Early Prophylaxis of Cytomegalovirus (CMV) Infection after Allogeneic Stem Cell Transplantation.
Authors: Bachier Carlos , Shaughnessy Paul , Wall Donna , Grimley Michael , Carrum George , Freytes Cesar , Callander Natalie , Walsh Tracey , LeMaistre C. Fred
Published: April 2020

Despite improvements in methods for the early diagnosis of CMV infection, 5% of HSCT patients receiving preemptive therapy develop CMV disease. In addition, the use of highly immunosuppressive therapies prior to and after transplant and the use of matched unrelated (MUD) and mismatched related (MRD) donors have increased the incidence of CMV infection and disease. We designed a prospective trial examining the safety and efficacy of valganciclovir for the prophylaxis of early CMV infection.

Methods: CMV seropositive patients or CMV seronegative patients receiving CMV seropositive stem cell products with an estimated creatinine clearance of ≥ 50 mL/min, platelet count ≥ 50,000/μL, and WBC count ≥ 1,000/μL are eligible. Patients receive valganciclovir 900 mg daily Mondays through Fridays starting 21 to 35 days after transplant and continuing through Day 100, with dosage adjustments for myelosuppression and/or reduced creatinine clearance. Patients are monitored with weekly CMV PCR analysis at a central lab according to the Roche assay.

Results: Twenty four (24) out of 50 planned subjects have been enrolled to date (MUD 9, MRD 15). All were CMV seropositive at the time of transplant. Stem cell source included peripheral blood = 21, bone marrow = 3. A total of 7 patients required corticosteroids for treatment of graft versus host disease. Three patients developed myeloid toxicity related to valganciclovir (absolute neutrophil count < 1,000/μL = 1, platelets < 50,000/μL = 2). All three (3) patients recovered their counts and restarted valganciclovir with dose adjustments. A total of 11 patients required dose adjustments due to myelosuppression (3), creatinine clearance (4), weight (1), institutional decision (3). Twelve (12) patients have not completed therapy due to: progressive disease or death before day +100 = 4, CMV infection =2, too early for analysis = 6. CMV infection occurred in 2/24 and CMV disease occurred in 0/24. The two (2) patients with positive CMV-PCR developed infection one (Day 40) and two (Day 36) weeks after starting valganciclovir. These two (2) patients were successfully treated with IV ganciclovir. Ten (10) patients have been followed between day +100 and 6 months post transplant. Three (3) of these 10 patients continued valganciclovir off study and had no evidence of CMV infection. Two (2) of the 10 patients who were not receiving valganciclovir developed CMV infection but were successfully treated with valganciclovir. Survival is 80% (16/20) at day +100 and 82% (9/11) at 6 months post transplant.

Conclusions: We conclude that valganciclovir at the dose used in this study is well tolerated with minimal reversible myelosuppression. These preliminary findings suggest that prophylaxis with oral valganciclovir appears to be a promising strategy to prevent CMV infection and disease.
Results of Hematopoietic Stem Cell Transplantation for Acute Myeloid Leukemia of the FAB M0 Subtype in First Complete Remission: A Survey of the Acute Leukemia Working Party of the European Bone Marrow Transplant Group (EBMT).
Authors: Herr Andree-Laure , Labopin Myriam , Reiffers Rosy , Bunjes Donald , Blaise Didier , Hurtenstein Bernard , Gorin Norbert C. , Gluckman Eliane , Rocha Vanderson
Published: April 2020

Hematopoietic stem cell transplantation (HSCT) is a potentially curative treatment for patients with acute myeloid leukemia (AML). AML of the FAB M0 subtype is rare, often associated with a complex karyotype and a poor prognosis. Results of HSCT for this AML subtype have never been reported separately from other subtypes. We did a survey of the results of 274 HSCT in adults with M0 AML in first complete remission (CR1), performed in EBMT centres since January 1990 until 2002. One hundred fifty patients were transplanted with an HLA identical donor (HLA-id), 30 with an HLA-matched unrelated donor (MUD) and 94 received an autologous transplant (auto). The median age was 45 years (16–71), the median interval from diagnosis to HSCT was 4 months for HLA-id, 6 months for MUD and 5 months for auto HSCT. The median follow-up time (range) was 20 months (1–109), 12 (2–53) and 10 months (1–96) for HLA-id, MUD and auto-HSCT respectively. The source of stem cells was peripheral blood stem cells for 67% of cases, and bone marrow for the remaining. The majority of grafts were non-T-cell depleted. Acute GVHD (grade I–IV) occurred in 56% of HLA-id and in 64% of MUD cases. The table shows the outcomes at two years according to the type of transplant. In conclusion, outcomes after HLA identical HSCT and MUD in adult patients with AML FAB subtype M0 in CR1 are encouraging. In comparison to allogeneic transplant cases, LFS is decreased in patients receiving an autologous transplant due to a high relapse incidence, reflecting the probable role of a graft-versus-leukemia effect in this FAB subtype.

Results of HSCT in AML M0 CR1 patients

Outcomes HLA-id n=150 MUD n= 30 Auto n=94 LFS: leukemia free survival; OS: overall survival; RI: relapse incidence; TRM: treatment-related mortality 2y LFS 50% 45% 33% 2y OS 58% 50% 49% 2y RI 25% 40% 57% 2y TRM 24% 14% 9%
Reduced-Intensity Conditioning for Allogeneic Hematopoietic Stem Cell Transplantation in Patients Older Than 60 Years.
Authors: Busca Alessandro , Locatelli Franco , Santarone Stella , Narni Franco , Morello Enrico , Rambaldi Alessandro , Mordini Nicola , Bruno Benedetto , Bacigalupo Andrea , Falda Michele
Published: April 2020

Allogeneic hematopoietic stem cell transplantation (HSCT) is an effective treatment for patients with hematological malignancies but its use is limited to young patients without comorbidities.

The aim of present study was to evaluate the efficacy of a reduced-intensity conditioning (RIC) consisting of fludarabine and TBI in older patients aged 60 to 70 years.

Methods: between March 2000 and May 2003, 22 patients (median age 62 years; range 59–70) with hematological malignancies (n=18 AML/MDS; n=1 ALL; n=1 CLL; n=1 myelofibrosis) and solid tumor (n=1 renal cell carcinoma) were treated with a RIC regimen based on fludarabine (30 mg/m2 x 3–5 days) and 200 cCy TBI followed by alloHSCT from a matched-sibling donor. GVHD prophylaxis consisted of cyclosporine and mycophenolate. The source of stem cell was blood in all patients. The median CD34+ content of the grafts was 5.7 x 106/kg (range 3.1–8.7). Three patients had received a prior allograft. Eleven patients had active disease at transplantation, 5 patients were untreated and 6 were allografted in complete remission (CR).

Results: neutrophil recovery occurred in 15 patients (68%) at a median time of 16 days (range 8–33). One patient who died early after transplantation (day +5) and 4 patients who experienced an autologous reconstitution, failed to reach this threshold; 2 patients did not become granulocytopenic. Fifty-eight percent of the patients had > 90% donor chimerism on day +30. Twelve of 17 evaluable patients (70%) developed acute GVHD (n=5 grade I; n=6 grade II; n=1 grade III), whereas chronic GVHD was observed in 11 of 13 patients (85%) surviving more than 100 days (limited in 7 cases and extensive in 4 cases). The probability of acute GVHD grades II–IV and extensive chronic GVHD was 43% and 31% respectively. Two patients died of treatment-related complications: 1 patient died of toxic encephalopathy on day 5 after transplantation and 1 patient of thrombotic thrombocytopenic purpura on day 915. The 100-day nonrelapse mortality for the whole population was 5%.Ten patients (45%) died as a result of relapse. With a median follow-up of 586 days (range 50–1128) 10 patients are alive (9 with CR and 1 with relapsed disease).The probability of overall survival at 2 years was 86% for patients treated in CR or at diagnosis and 9% for patients transplanted in advanced phase of disease (p= 0.0008).

Conclusion: Our analysis would suggest that for patients older than 60, this RIC regimen is well tolerated and associated with a low incidence of transplant-related mortality and serious acute and chronic GVHD. The leukemic burden at time of transplant has proven to be the most important risk factor for the outcome.
Economic Analysis of Reduced-Intensity Cord Blood Transplantation.
Authors: Yuji Koichiro , Miyakoshi Shigesaburo , Myojo Tomohiro , Miura Yuji , Kato Daisuke , Imataki Osamu , Narimatsu Hiroto , Kusumi Eiji , Matsumura Tomoko , Kami Masahiro , Masuoka Kazuhiro , Ueyama Junichi , Wake Atsushi , Morinaga Shinichi , Taniguchi Shuichi
Published: April 2020

[Background] Non-myeloablative regimens have been proven to allow engraftment following allogeneic stem cells transplantation with minimal procedure-related toxicity and lower costs. Cord blood has emerged as an appealing alternative source of hematopoietic stem cells for unrelated donor transplantation, but delayed engraftment and frequent transfusion were reported. No studies have formally evaluated the cost of reduced-intensity cord blood transplantation (RICBT).

[Purpose] To evaluate the relationship among costs, baseline patient characteristics, and major complications of RICBT, we performed an economic analysis of data in a clinical trial of RICBT for hematologic diseases at a single institution.

[Patients and Methods] Ninety-three patients with hematological diseases (median age, 55y; range, 17–79: median body weight, 53kg; range, 38–75) underwent RICBT from March 2002 to May 2004 in Toranomon Hospital. Mean follow-up period was 77 days (range, 13–863). Data on resource use, including hospitalizations, medical procedures, medications, and diagnostic tests, were abstracted from subjects’ clinical trial records. Resources were valued using the Japanese national insurance reimbursement system for inpatient costs at one hospital and average wholesale prices for medications. Monthly costs were calculated and stratified by treatment group and clinical phase.

[Results] The median initial inpatient cost was $80,400 (range, 41,300–154,700). When baseline variables were considered, disease status was significant predictor of costs. When clinical events were considered, in-hospital death was associated with higher costs. The mean length of total inpatient days was 78 days (range, 31–222), and the mean length of inpatient days post transplant was 51 days (range, 15–131). The mean units of transfused RBC, Platelet, and FFP were 27u, 224u, and 27u, respectively.

[Discussion] This study firstly demonstrates that the cost of RICBT was much higher as compared to previous RIST using peripheral blood or bone marrow. RICBT is an attractive therapy, however, economic problem lies before prevalence of RICBT. The increased numbers of transfusions and supportive care would have effects on costs. The association between mortality and higher costs suggest that prevention of clinical complication may have significant economic benefits. Interventions that decrease these complications may have favorable cost-benefit ratios, and will be the focus of future investigation.
mTOR as Therapeutic Target for T-Cell Leukemia: Synergism with Conventional Cytotoxic Drugs and Signaling Inhibitors.
Authors: Batista Ana , Barata Joao T. , Costa Lara F. , Sallan Stephen E. , Carlesso Nadia , Nadler Lee M. , Cardoso Angelo A.
Published: April 2020

Targeted disruption of signaling pathways essential for tumor cell survival and proliferation offer great promise for the design of more efficient and specific therapies. mTOR plays a central role in sensing mitogenic, nutrient and energy signals, and in the integration of different signaling pathways. Moreover, increasing evidence implicates mTOR in tumorigenesis and in the maintenance of transformed phenotypes. Therefore, we hypothesized that the mTOR pathway may play a significant role in T-cell acute lymphoblastic leukemia (ALL). BM and peripheral blood specimens from 15 pediatric patients were analyzed for the phosphorylation status of two downstream substrates of mTOR, S6 Ribosomal Protein and eIF4G. In all cases, primary leukemia T cells showed marked phosphorylation of S6 Ribosomal Protein (Ser235/236) and phosphorylation of the eukaryotic initiation factor eIF4G (Ser1108), showing that the mTOR pathway is constitutively active in T-ALL cells. Since leukemia T cells did not show significant basal phosphorylation of Akt/PKB, this observation suggests that the mTOR activation observed in T-ALL is Akt-independent. Specific blockade of mTOR by Rapamycin (100nM) reduced or abrogated phosphorylation of S6 ribosomal protein. Moreover, the mTOR blockers Rapamycin (10–100nM) and CCI-779 (10–100nM) significantly inhibited cell survival, and IL-7-promoted proliferation (40% to 85% inhibition) and cell cycle progression of T-ALL cells. Since we have shown previously that PI3K/Akt is critically involved in T-ALL responses to exogenous stimuli, we next evaluated whether inhibition of PI3K/Akt signals would synergize or potentiate the effects of mTOR blockade. We observed that the PI3K-inhibitor LY294002 (10μM) synergized with Rapamycin to abrogate IL-7-promoted leukemia T-cell proliferation. Finally, we explored the possibility that mTOR blockade can enhance the cytotoxic effects on T-ALL of the conventional drugs Dexamethasone (100nM) and Doxorubicin (100nM). Rapamycin potentiated the cytotoxic effects of both Dexamethasone (1.3 to 10 fold) and Doxorubicin (1.7 to 7.3 fold), suggesting that the combination of mTOR blockade with chemotherapy can improve the anti-leukemia efficacy of conventional regimens. In conclusion, we observed that the mTOR pathway is constitutively activated in primary leukemia T cells, and that the mTOR blockers Rapamycin and CCI-779 significantly inhibit T-ALL survival and proliferation. In addition, Rapamycin potentiates the inhibitory activity of other signaling inhibitors and the cytotoxic effects of the conventional drugs Dexamethasone and Doxorubicin. This study suggests that the mTOR pathway is a valid target for the treatment of T-ALL and lay the groundwork for the inclusion of mTOR blockade in the current therapeutic regimens.
The Survival of Multiple Myeloma Patients: A Single Institution Study.
Authors: Berneman Zwi N. , Verstraete An-Sofie , Gadisseur Alain , Van de Velde Ann , Schroyens Wilfried A.
Published: April 2020

Background: For a long time, multiple myeloma has been a disease with a poor outcome. High dose (melphalan) chemotherapy followed by autologous stem cell transplantation has been reported to improve the overall and progression-free survival of these patients.

Objective: To determine the survival of multiple myeloma patients treated with conventional chemotherapy and compare it with that of patients treated with high-dose chemotherapy followed by autologous peripheral blood stem cell transplantation.

Design/Methods: 83 myeloma patients treated at a single institution were included in this retrospective study. They were divided into two groups: one group of patients who were received high-dose chemotherapy followed by autologous peripheral blood stem cell transplantation (n=42) and one group of patients who only received conventional chemotherapy and were eventually also treated with thalidomide and/or corticosteroids (n=41). The distribution of the stages of the disease according to Salmon and Durie were similar in both groups of patients. For both groups, the overall and progression-free survival was calculated.

Results: In the general analysis, myeloma patients who underwent an autologous transplant had a significantly longer overall survival (58.8 vs. 52.2 months, p=0.036) and progression-free survival (39.6 vs. 11.8 months, p < 0.001) in comparison with the conventional chemotherapy group. If analysis was restricted to those patients who were transplanted as a first-line treatment, there was no significant difference in overall survival in comparison with conventional chemotherapy (51.8 vs. 52.2 months, p= 0.422); progression-free survival was significantly better in the first-line transplant arm as compared to the conventional chemotherapy arm (35.4 vs. 11.8 months, p= 0.003). As the median age in the transplant arm was significantly lower than in the conventional chemotherapy arm, we also performed a sub-analysis of patients who were between 60 and 70 years of age at diagnosis; there was no significant difference in overall survival between the two groups (60.7 vs. 69.5 months, p= 0.656), while the progression-free survival was again better in the autologous transplant group as compared to the conventional chemotherapy group (41.0 vs. 8.4 months, p= 0.020).

Conclusion: High-dose chemotherapy and autologous stem cell transplantation in the treatment of myeloma is associated with improved progression-free survival and in the general analysis, with improved overall survival. The overall survival of patients who were only treated with conventional chemotherapy is somewhat higher (more than 4 years) as compared to that of historical controls (2–3 years).
Retroviral Gene Transfer with Triple Genetic Reporter Genes into Human Cord Blood CD133+ Cells.
Authors: Niu Ting , Najjar Amer M. , Robinson Simon N. , Yang Hong , Decker William K. , McMannis John D. , Ng Jingjing , Thomas Michael W. , Champlin Richard E. , Gelovani Juri G. , Shpall Elizabeth J.
Published: April 2020

Umbilical cord blood (CB) is used increasingly as a source of hematopoietic support in bone marrow transplant patients lacking family or unrelated donors. Little is known about human CB with respect to in vivo migration, homing, long-term viability, the organ-specific checkpoints and its differentiation. In order to study CB migration and homing, the gibbon ape leukemia virus (GALV)-pseudotyped retroviral gene transfer technique was developed to genetically modify CB CD133+ cells with triple reporter genes which encode enhanced green fluorescent protein (eGFP), luciferase, and herpes simplex virus type one thymidine kinase (HSV1-tk). The novelty of this approach is that it allows multimodal repetitive non-invasive imaging using fluorescence, bioluminescence (BLI), and nuclear imaging techniques, respectively. CD133+ cell fractions were isolated from human CB using the MidiMACS device (Miltenyi). To facilitate transduction, CB CD133+ cells were stimulated with 100ng/ml each of stem cell factor (SCF), granulocyte colony-stimulating factor (G-CSF) and thrombopoietin (TPO) in alpha-MEM medium supplemented with 20% fetal bovine serum. After 3, 5, 7 or 14 days of stimulation, CB cells were transduced by RetroNectin (TaKaRa)-assisted gene transfer. RetroNectin, recombinant human fibronectin fragment CH-296, can provide colocalization of retroviral particles and target cells. Specifically, 6-well plates were coated with 5ug/cm2 RetroNectin for 2 hours at room temperature, followed by preloading with fresh virus-containing medium (VCM) for 4 hours at 37°C. VCM was then removed and the CB cells were added in medium containing cytokines for 2 or 3 days. This approach resulted in > 90% transduction of control U87 human glioma cells and up to 36% transduction of CD133+ CB cells. Preliminary experiments suggested that optimal transduction appeared to be at day 7 of CB stimulation. When compared to non-RetroNectin coated plates, this approach showed a 5- to 11- fold increase in eGFP+ cells as determined by flow cytometry, increasing the transduction efficiency from < 5% to a maximal 36%. The addition of polybrene, a cationic polymer, to this process did not improve transduction efficiency. Studies are underway to visualize retrovirally-transduced CB CD133+ cells in NOD/SCID mice in vivo. The ability to trace the fate of CD133+ cells following transplantation may allow a better understanding of the mechanisms of migration, homing and long-term viability.
Intratumoral, Injection of Adenoviral Flt3 Ligand Has Therapeutic Activity in Association with Increased Intratumoral Levels of T Cells but Not Dendritic Cells.
Authors: Talmadge James E. , Kurz Scott G. , Fields Randy R. , Agrawal Devendra K. , Singh Rakesh K. , LaFace Drake M. , Ashour Abdel-kader E. , Shafer Lora R. , Solhwim Joyce E.
Published: April 2020

Fms-like tyrosine kinase 3 ligand (Flt3L) is a growth factor for dendritic cells (DCs), hematopoietic stem cells, and natural killer (NK) cells in vivo. Ten daily injections of Flt3L, systemically expands DCs, whereas a single injection of an adenovirus vector with the Flt3L transgene (Adv-Flt3L) significantly expands DCs as soon as 2 days following intravenous (iv) injection. The increases in DC numbers, following iv injection, remains at near peak levels from day 6 through day 16. Maximal DC expansion occurs following the iv injection of 1011 virus particles, such that eight days following the iv injection of Adv-Flt3L it induced an 8 fold increase in splenic CD11c+CD11b−CD8a+ cells (immune augmenting DCs), a 6 fold increase in splenic CD11c+B220+ plasmacytoid DCs, and a 9 fold increase in splenic CD11c+CD11b+ cells (immunosuppressive DCs). The increase in DC numbers and cellularity returns to near normal levels by 22 days following iv injection of Adv-Flt3L. Despite the 2–3 fold increase in spleen cellularity and significant expansion of DCs in the spleen, blood and lungs by the iv injection of Adv-Flt3L, this route of administration has minimal to no therapeutic activity for orthrotopic mammary tumors, even when therapy is initiated against small primary tumors. In contrast, the intratumoral injection of Adv-Flt3L has significant therapeutic activity against orthrotopic mammary tumors including mice in which therapy was initiated against large tumors. Unexpectedly, no significant increase in DC infiltration of the tumor was observed following iv or intratumoral Adv-Flt3L administration. In contrast, the number of tumor infiltrating CD4+ and CD8+ cells was increased following intratumoral but not iv Adv-Flt3L administration, suggesting an association with therapeutic activity. Adv-Flt3L injection also induced DC expansion in the spleen, PB and lungs in cl-66, mammary adenocarcinoma-bearing mice. Injection of adv-Flt3L (iv or intratumoral) also stimulated the expansion of T-cells (both CD4+ and CD8+) and a type 1 T cell response as measured by qRT-PCR. Further, the therapeutic activity and increased mitogenic responses by splenic lymphoid cells to Con-A and IL-2 was not depressed in mice bearing cl-66 tumors and the effect of Adv-Flt3L, while partially associated with the Adv vector, was significantly higher in mice injected with Adv-Flt3L.
Conjugation of Plasminogen Activators to Carrier Erythrocytes Imparts New Biological Properties to Their Soluble Counterparts.
Authors: Ganguly Kumkum , Krasik Tatiana , Bdeir Khalil , Cines Douglas B. , Muzykantov Vladimir R. , Murciano JuanCarlos
Published: April 2020

Conjugation of plasminogen activators (PA) to carrier red blood cells (RBC) generates a new agent (RBC/PA), which selectively lyses nascent blood clots. In this work, we studied two types of RBC/PA conjugates, carrying Alteplase (tissue type plasminogen activator, tPA) or Reteplase (an engineered form of tPA, Ret). Compared to non-conjugated 125I-PA counterparts, both 125I-tPA and 125I-Ret coupled to 51Cr-RBC via biocompatible biotin-streptavidin cross-linker showed markedly prolonged circulation in rats and mice. Despite slightly faster blood clearance, RBC/tPA retained significantly higher fibrinolytic activity in circulation than RBC/Ret. In part, the higher fibrinolytic activity of RBC/tPA vs RBC/Ret in the circulation was due to its lessened susceptibility to plasma inhibitors. Analysis of amidolytic activity of RBC-coupled vs free tPA and Ret using chromogenic substrates in vitro revealed that coupling to RBC rendered Ret, but not tPA, insensitive to stimulation of fibrinolytic activity by fibrin. In vitro binding assay in cultural wells showed that RBC/tPA specifically binds to fibrin clot (6±0.2x104 RBC/tPA bound per well vs. 1±0.4x104 RBC/Ret or 7.3±0.6x103 naive RBC). RBC/tPA also bind specifically to immobilized fibrinogen and plasminogen (8±2x104 and 2±0.4x104 RBC/well), but not to non-cleavable plasminogen, collagen, fibronectin or thrombospondin (<1.1±0.3x103RBC/well). Neither RBC/Ret nor naïve RBC bind to these proteins (<1.1±0.2x103 RBC/well). Therefore, RBC/PA complexes display a considerable functional diversity, a result of interplay between the pharmacokinetic features offered by carrier RBC, individual features of a given PA and alterations of the latter caused by RBC coupling. In theory, this diversity may enhance flexibility and utility of potential applications of RBC/PA for thromboprophylaxis.
IL-6 and Anemia of Inflammation.
Authors: Rivera Seth , Gabayan Victoria , Ganz Tomas
Published: April 2020

BACKGROUND: Anemia of inflammation (AI, anemia of chronic disease) is an iron-restricted anemia caused by hepcidin limiting the supply of iron for erythropoiesis. We had previously shown that IL-6 was necessary for the induction of hepcidin and the development of hypoferremia during acute inflammation in vivo (Nemeth E et al, JCI, 2004). In vitro, IL-1 (Lee P et al, PNAS, 2005) and TGF-β (unpublished data) can also stimulate hepcidin production in primary murine hepatocytes in an IL-6 independent fashion.

METHODS: We sought to determine whether IL-6 was necessary for the development of AI using a model of chronic peritonitis in IL-6 deficient mice and wild type (WT) controls.

RESULTS: IL-6 deficient mice developed anemia of similar severity as their WT counterparts. WT mice developed microcytic anemia consistent with inflammation-induced iron restriction. In contrast, the inflamed IL-6 deficient mice developed mild macrocytosis indicating markedly different erythrocyte iron metabolism. Hepcidin mRNA increased in WT mice but not IL-6 deficient mice in response to inflammation.

CONCLUSION: IL-6 is the principal regulator of hepcidin during acute and chronic inflammation. However, anemia still occurs in the absence of IL-6. The difference in mean corpuscular volume implies that the mechanism of anemia in IL-6 deficient mice is likely different than in WT mice.