bluuebunny/crossref_metadata_2025 huggingface dataset:
Reduced Intensity Conditioning Regimen for Allografting Following Autografting Has Strong Anti-Myeloma Activity and Delays Disease Progression Compared to Tandem Autografting.
Authors: Carella A.M. , Lerma E. , Nati S. , Congiu A. , Rossi E. , Spriano M. , Vimercati R.
Published: April 2020

Multiple Myeloma (MM) is still an incurable disorder despite the numerous attempts to exploit new therapy approaches for it. The better understanding of its molecular pathogenesis has led to the development of effective, novel therapeutic agents like thalidomide and the proteasome inhibitor bortezomib. High-dose therapy with stem cell transplantation and novel targeted therapies represent two approaches to overcome resistance of multiple myeloma cells to conventional treatments. Autografting (AutoSCT) has been limited by high-relapse rates and conventional allografting (AlloSCT) by excessive TRM and toxicity. Reduced intensity conditioning for transplant (RICT), a less toxic procedure for AlloSCT that aims to exploit graft versus tumor effect, has been shown to achieve remissions in MM. High-dose therapy/AutoSCT followed shortly thereafter by RICT might improve outcomes in MM as compared to AutoSCT or conventional AlloSCT used alone. We compared two retrospective cohort of patients who underwent either tandem AutoSCT (HDT consisted of Melphalan 200 mg/m2) or AutoSCT followed closely by related RICT (patients with HLA-matched siblings). The two groups were matched for pre-transplant therapy, disease status at transplant, time from diagnosis to transplant. GVHD prophylaxis for RICT patients consisted of CyA/MTX. The major results are summarized in the Table. In the AutoSCT/RICT group the complete remission rate was higher (p=0.004) and the risk of disease progression after transplant was significantly reduced. All patients who reached CR responded after full chimerism and GVHD developed. This finding confirms the existence of a graft-versus-myeloma effect. Since the first clinical signs of response in remitters patients were noted between 70 and 120 days and maximum response between 160 and 200 days after RICT (after DLI in one patient), these responses should be considered immunological responses. Our data demonstrated that RICT following an AutoSCT can mediate a potentially curative graft-versus-myeloma effect and reduces the incidence of disease progression.

Tandem ASCT (N=35) ASCT+RICT (n=20) Age, median 56 (range, 38–66) 51 (range, 34–63 Median number of prior cycles of Chemoth. 4 (range, 3–6) 4 (range, 3–6) Time from Dx to 1st AutoSCT (median mo.) 6 (range, 5–60) 9 (range, 7–42) Conditioning Regimen for AutsoSCT Melphalan (200mg/m2) Melphalan (200mg/m2) Conditioning regimen for RICT --- TBI/Fludarabine Median days from AutoSCT to RICT --- 80 Complete Remission rate 14% 50% Disease-free Survival at 4 yrs 14% 45% Overall Survival at 4 yrs 28% 40% Transplant-Related Mortality 0% 0%
Clinical CMRF-56 Blood Dendritic Cell Preparations for Immunotherapy of Multiple Myeloma.
Authors: Vari Frank , Rossetti Tony , Freeman Jennifer , Hart Derek
Published: April 2020

Aim: Despite advances in therapy, such as stem cell transplantation and new drugs such as the proteosome inhibitors, multiple myeloma (MM) remains incurable for most patients. Immune therapy, which uses dendritic cells (DC) to generate cytotoxic T lymphocyte (CTL) responses targeting residual disease is a promising, potentially curative strategy, compatible with other therapies. We have shown that blood DC can be isolated from MM patients and that they induce CTL responses (J. Immunotherapy.2005; 28:322). The CMRF-56 monoclonal antibody (mAb) detects an antigen expressed on blood DC after a short period of in vitro culture. To create a GMP compatible DC purification platform for clinical investigation of DC immunotherapy, we developed a method for single step CMRF-56 mAb positive immunoselection of blood DC using magnetic beads.

Methods and Results: CMRF-56 mAb immunoselection produced a blood DC enriched population containing 33.2±3.5% CD11c+ blood DC (including CD1c+, CD141+ and CD16+ subsets) and 0.6±0.2% CD123hi plasmacytoid blood DC (n=6). Additional CMRF-56 positive B cells and monocytes comprised 14.7±5.8% and 32.4±3.4% of the preparations respectively (n=6). CMRF-56 selected cells stimulated CTL responses against defined tumour associated antigens, including MART-1 peptide and the MM associated antigen, HM1.24. Furthermore they generated strong MM specific CTL responses after incubation with MM cell lysates. Brief DC activation with GM-CSF enhanced these responses, improving MM-specific CTL activity (Cr release) in cultures primed with MM lysate loaded CMRF-56 selected cells from 18.9±7.4% to 44.1±8.0% (n=3, p=0.051). We have now developed and validated a procedure for isolation of CMRF-56+ blood DC using the CliniMACS device. Apheresed mononuclear cell preparations were cultured, labelled with antibody and blood DC immunoselected in a closed system, free of human or animal serum supplements, in a manner compatible with regulatory requirements for cell therapy. Simultaneously, our team has developed a bioprocess for production of the murine biotinylated CMRF-56 mAb, suitable for the large scale synthesis required for multi-centre clinical trials. It incorporates components that remove contaminating DNA, host cell proteins, viral particles and endotoxin. The first batch of clinical grade CMRF-56 mAb has been produced and used for successful laboratory scale blood DC isolation. We have also completed a number of clinical scale blood DC preparations from healthy donors (n=10) and MM patients (n=2). There were sufficient blood DC isolated from both the healthy donors and MM patients for the planned vaccination schedule. The average blood DC purity was 38% in healthy donors and 29% in MM patients, while the average yields were 35 and 44%, respectively.

Conclusions: When additional studies on MM patients are completed these studies will provide the necessary pre-clinical validation data to commence a Phase I Clinical Trial.
Erythropoietic Agents and Venous Thromboembolic Events in Multiple Myeloma Patients Treated with Anthracycline Based Chemotherapy and Immunomodulators.
Authors: Baz Rachid , Fruchtman Steven M. , Zeldis Jerome B. , Walker Esteban , Hussein Mohamad A.
Published: April 2020

Background: Erythropoietic agents (EPO) have recently been implicated with higher rates of thromboembolic events (VTE) in multiple myeloma (MM) patients receiving lenalidomide (R) and dexamethasone (Knight et al. NEJM 2006). Uncontrolled erythrocytosis, a recognized risk factor for VTE, may complicate therapy with EPO, but was not evaluated as a causative factor for VTE in the above report. We sought to evaluate the putative interactions between erythrocytosis, immunomodulator therapy, EPO and VTE.

Patients and Methods: We retrospectively reviewed patients’ records from 2 studies of anthracycline-based chemotherapy with immunomodulators: pegylated liposomal doxorubicin, vincristine, dexamethasone and thalidomide (T) (study A) or lenalidomide (study B) in MM. Demographic variables, aspirin therapy, EPO therapy, immunomodulator therapy (T vs R), as well as laboratory variables (which included all hemoglobin values while on study) were reviewed.

Results: From August 2001 to December 2005, 184 multiple myeloma patients with active disease were treated on study A (105) and B (79). The median age for patients was 63 and 62 years on study A and B respectively. Aspirin prophylaxis (81mg daily) was administered for all patients on study B and for all but 19 patients on study A. Among all patients, 113 patients (61%) received EPO. Twenty-six patients on study A and 7 patients on study B developed a VTE. Treatment with EPO was not associated with a higher rate of VTE compared to no treatment with EPO (19% versus 22%, Fisher exact test, p=0.8). The mean peak hemoglobin for patients with a VTE was not statistically different from patients without a VTE (14.2 vs. 13.8 g/dL, p=0.3). Similarly, the mean on study hemoglobin was not different among patients with a VTE and those without a VTE (11.7 vs. 11.6 g/dL, p=0.8). The mean hemoglobin at the time of the VTE was 12.0 g/dL (S.D. 1.5g/dL).

Conclusion: Our results do not indicate an association between EPO and VTE in MM patients receiving anthracycline-based chemotherapy with an immunomodulator and aspirin prophylaxis. Uncontrolled erythrocytosis does not appear to be associated with VTE in this patient population. Of note, target hemoglobins were not commonly exceeded. Low dose Aspirin (81mg) is an effective prophylaxis for VTE in multiple myeloma patients receiving anthracycline based therapy with thalidomide or lenalidomide in conjunction with the use of erythropoietin therapy.
Osteonecrosis of the Jaw (ONJ) in Patients with Multiple Myeloma, Receiving Bisphosphonate Therapy, Is Independent of Renal Function: Single Institute Experience of an Emerging Clinical Problem.
Authors: Manochakian Rami , Coignet Jean-Gabriel , Padmanabhan Swaminathan , Sullivan Maureen , Miller Kena C. , Chanan-Khan Asher Alban
Published: April 2020

Introduction: Use of bisphosphonate (zoledronic acid and pamidronate) for prevention of pathological fractures is standard of care in multiple myeloma (MM) patients with osteolytic bone lesions. While clinical trials have investigated effectiveness of these agents for up to 2 years of use, clinical practice has incorporated their use indefinitely. With improvement in survival through availability of novel agents in MM patients, the use of bisphosphonate therapy has also prolonged. Recent reports unveiled a new side effect of Bisphosphonate therapy i.e., ONJ, characterized by necrosis of the jawbone with antecedent subluxation or loss of the teeth and ulceration of the gum mucosa. The underlying etiology remains elusive, and in the absence of effective remedy ONJ has raised significant concerns upon the duration of treatment with Bisphosphonate. We investigated the magnitude of this problem among MM patients receiving Biphosphonate therapy at our institute.

Methods: We conducted a retrospective review of all patients diagnosed with MM at our institute since 2001 who received bisphosphonate treatment and developed ONJ. Clinical characteristics, prior treatments and renal function prior to development of ONJ were evaluated.

Results: Records of 20 pts who developed ONJ were reviewed.

The median age of these pts was 57 (range 45–69).

Among these, 9 were males, 11 were females. All pts (100%) had stage III MM.

11 patients (55%) received 2 or more antimyeloma treatment.

14 pts (70%) were on Zoledronic acid, 5 pts (25%) were on Pamidronate, and 1 pt (5%) was first on Zoledronic acid then switched to Pamidronate.

Out of those 20 patients, 19 (95%) had normal renal function prior to and after development of ONJ, with Creatinine level range of (0.5–1). Only 1 patient (5%) had decreased renal function prior to development of ONJ, with Creatinine level of 6.6

Conclusion: Although the number of patients reported are small, experience at our center with ONJ in MM patients who were on Bisphosphonate failed to show any association with renal function. Interestingly all patients who developed ONJ were stage III MM.

There are currently no established guidelines for prevention or treatment of this disorder.
Effect of Chemotherapy Treatment on Fetal-Maternal Microchimerism in Parous Cancer Patients.
Authors: Gilmore Gary L. , Holm Melissa M. , Anikanova Yuliya , Haq Bushra , Jasthy Sri Lakshmi , Lister John , Rossetti James M. , Shadduck Richard K.
Published: April 2020

Fetal-maternal microchimerism [MC] is a benign condition arising from cross-trafficking of circulating cells between the fetus and the mother during gestation. It is known that MC persists for decades post-partum. The reported incidence of MC is 33% in normal parous women, but the MC status of cancer patients has not been examined. We have completed our study of 200 parous cancer patients, using a sensitive two-round PCR technique with nested primer sets specific for the Y-chromosome specific sequence, DSY14, to detect male DNA in blood samples from female patients.

Exhaustive analysis of samples from parous cancer patients gave a frequency of 34% MC+ [68 of 200], which is significantly lower than the 57% MC+ [114 of 200] we found in normal parous women [p<.0001]. We reported an apparent dichotomy based on diagnosis, in that patients with hematologic malignancies had a greater frequency of MC than solid tumor patients [46% vs 29%; p<.0001]. Both groups received similar numbers of chemotherapy cycles [3.4 cycles for solid tumor patients; 3.2 for hem/onc patients], suggesting this difference was not due to less intensive treatment.

When we separated the untreated patients from each population, we found that only 25% [3 of 12] untreated patients with hematologic malignancies were MC, whereas 32% of untreated solid tumor patients were MC+ [12 of 38; p=.66, ns]. Therapy appeared to increase the frequency of MC in hematologic patients to 52% [25 of 48; p=.09, ns], but MC was essentially unaffected in solid tumor patients [27%, 28 of 104; p = .18, ns]. The difference between treated solid tumor patients and those with hematologic malignancies was significant [p=.0001]. Thus, one side effect of chemotherapy may be to increase MC in hematologic malignancy, but in solid tumor patients, MC is not affected.

The reason for the increased MC in treated hematologic patients in unclear, but we speculate the reduced frequency in untreated patients is due to dilution by the malignant clone, and that chemotherapy restores the normal balance of blood cells in these patients. The reason for reduced MC in solid tumor patients is also not obvious, as DNA yields are roughly equivalent between the two groups of cancer patients. Further analysis of the data is underway to see if an underlying cause can be determined.
The Balance in Chimerism between T Cells and Blood Dendritic Cells in Relapsed CML Patients Influences the Induction of Alloreactivity Following Donor Lymphocyte Infusion.
Authors: Levenga Henriette , Woestenenk Rob , Schattenberg Antonius V.M.B. , Maas Frans , Jansen Joop H. , Raymakers Reinier , De Mulder Pieter H.M. , Schaap Nicolaas , de Witte T.M. , Dolstra Harry
Published: April 2020

Donor lymphocyte infusions (DLI) after allogeneic stem cell transplantation induce complete remissions in 70 to 80% of patients with relapsed CML in chronic phase, but some patients do not respond sufficiently to DLI. We studied chimerism in subsets of immune cells in relation to the induction of alloreactivity. T cells and two subsets of DC (blood precursor myeloid dendritic cells [MDC] and blood precursor plasmacytoid dendritic cells [PDC]) were isolated from 15 relapsed CML-patients shortly before DLI for chimerism analysis. Furthermore, the absolute blood counts of DC-subsets were determined. Based on chimerism we identified three groups. Group 1 (4 patients) was complete donor chimeric in T cells and DC-subsets. These patients had an early stage of relapse and 3 of 4 patients attained a complete molecular remission (mCR) without significant GVHD. Group 2 (6 patients) was complete donor in T cells and mixed chimeric in DC-subsets. Median percentage of recipient MDC in 4 patients with mixed chimerism was 67% (range, 36%–72%). One patient was complete donor and one patient complete recipient chimeric in MDC. Median percentage of recipient PDC was 37% (range, 18%–58%) in 5 patients with mixed chimerism and in 1 patient PDC were complete recipient derived. All patients entered mCR, however in combination with GVHD in 4 and bone marrow hypoplasia in 3 patients. Group 3 (5 patients) had mixed chimerism in T cells and complete recipient chimerism in MDC in 4 of 5 patients. Only 2 patients entered mCR. Absolute DC numbers at the time of DLI did not predict the induction of an allo-immune response, however very low numbers of MDC and PDC were associated with progressive disease and a poor outcome. The combination of donor chimerism in T cells and mixed chimerism in DC-subsets in advanced relapse is associated with the most potent GVL effect following DLI. GVL and GVHD are separated in patients with an early relapse and donor chimerism in both T cells and DC-subsets. Finally, absolute DC numbers do not predict the strength of the alloresponse.
The Role of CD4+CD25+ T Cell and FOXP3 in Acute Graft Versus Host Disease.
Authors: Liu Yue-jun , Wu De Pei , Li Cai-xia , He Jun , Qiu Qiao-cheng
Published: April 2020

Objective To explore the relationship among CD4+CD25+ regulatory T cell, the expression of FOXP3 mRNA levels of donor bone marrow (BM) and graft versus host disease (GVHD) in allogeneic hematopoietic stem cell transplantation (allo-HSCT).

Methods

Thirty patients undergoing allo-HSCT were enrolled in this study. The levels of donor CD4+CD25+ regulatory T cell were compared in the patients with GVHD and those without GVHD by immnofluorescence and flowcytometry. The donor BM expressions of Foxp3 mRNA levels were analyzed by using reverse transcriptase polymerase chain reaction (RT-PCR).

Results

All patients achieved engraftment. ANC ≥0.5×109/L and PLT ≥ 20×109/L were at day 14(range, 12–15)and day 18 (range, 15–25) respectively. With a median follow-up of 12.8 (8–16) months, Acute GVHD occurred in 15 of 30 patients(50.0%) with gradeII–IV Acute GVHD (40.0%). Chronic GVHD developed in 6 (extensive 2, limited 4) out of 30 patients (20.0%). The levels of donor CD4+CD25+ regulatory T cell pre-and pro-mobilization were (2.67±0.38)% and (5.01±1.33)% respectively, no difference were observed (P>0.05). Patients with I–II aGVHD demonstrated higher donor-type CD4+CD25+ immune regulatory T cell level than those with III–IV aGVHD; Patients with III–IV aGVHD showed much lower donor-type CD4+CD25+ immune regulatory T cell level than those without GVHD; no difference were observed between patients withI–II aGVHD and those without aGVHD. The transcripts of FOXP3 donor BM were detected in seven donors of thirty recipients who received allo-HSCT. Five patients had no GVHD after transplantation;two developedIaGVHD. Donor BM Foxp3 transcripts were never detected in patients with II–IV aGVHD.

Conclusion

Donor-type CD4+CD25+ immune regulatory T cell level was relative to recipient II–IV aGVHD. Up-grade donor-type CD4+CD25+ immune regulatory T cell level could reduce the incidence of aGVHD. Donor BM FOXP3 transcripts were detected in patients without serious aGVHD after transplantation.
Enhanced Human Hematopoietic Cell Engraftment and T Lymphocyte Development in NOD/Scid/Jak-3 Null Mice.
Authors: Okada Seiji , Harada Hideki , Suzu Shinya
Published: April 2020

Xenotransplantation of human cells into immunodeficient mice has been used to develop models of human hematopoiesis and immune function. In this study, we generated the NOD/Scid mice with null mutation of Jak-3 (NOD/Scid/Jak-3 null) by 10 backcross mating of C57/BL6-Jak-3 null mice and NOD/Scid mice. NOD/Scid/Jak-3 null mice had NK and NKT cells as well as mature T and B lymphocytes. NK activity was not also detected in this mice.

Intrahepatic injection of CD34+ human cord blood cells into irradiated (2.0Gy) newborn NOD/Scid/Jak-3 null mice led to de novo development of B and T lymphocytes. In addition to the high engraftment rate of human hematopoietic cells, multilineage cell differentiation including T cell lineage was obserbed. T lymphocyte appeared in peripheral blood as early as 8 weeks after transplantation and it was increased in number thereafter. Both naive and memory phenotype CD4 and CD8 T lymphocytes were detected 12 weeks after transplantation. Plasma cells were detected in the bone marrow 20 weeks after transplantation.

Furthermore the engrafted mice were infected with HIV. Our model mice provide a valuable model to study not only development and function of human hematopoeitic and immune system but also the immune response to the cancer and infectious disease such as HIV.
Rapid Generation of Cytomegalovirus Specific T Lymphocytes Using Commercially Available CMV pp65 Peptides.
Authors: Lucas Kenneth G. , Sun Qi , Niyazova-Brewer Nargisa , Bao Lei
Published: April 2020

Adoptive immunotherapy with cytomegalovirus (CMV) specific cytotoxic T lymphocytes (CTL) can result in resolution of viremia and reconstiution of CMV specific immunity following allogeneic stem cell transplantation. Most recipients of allogeneic transplants who develop CMV reactivation are successfully treated with anti-viral agents, however some have persistent viremia or develop CMV disease despite these medications. In these situations the development of CMV specific T cells may be impractical due to time contraints or institutional limitations. We have expanded CMV pp65 specific CTL from 5/6 normal blood donors of diverse HLA backgrounds in 10–15 days using adherent cells (monocytes) as antigen presenting cells, pulsed with a commercially available source of CMV pp65 peptides, which includes 138 CMV pp65 15mers. These pulsed monocytes are incubated at a 1:5 ratio with non-adherent peripheral blood lymphocytes, resulting in a population of CD4 and CD8+ CMV pp65 specific T cells after 2 weekly stimulations. While >80% of these cells were CD3+, 29–78% were CD4+, and 15–55% were CD8+. These CTL have specific cytotoxicity against CMV pp65, and intracellular cytokine analysis showed specific interferon-gamma production in response to CMV pp65 target cells. Table I depicts the phenotype as well as interferon-gamma production and specific cytotoxicity in response to stimulation with B lymphoblastoid cell lines (BLCL) expressing pp65. There was no reactivity to allogeneic targets by cytokine analysis or in chromium release assays. CTL sufficient for a dose of 30–50 × 106 CTL were routinely generated from 40–50 cc of whole blood. Considering the short time frame and relative ease with which these CTL can be expanded, this methodology could be readily adopted in clinical situations of persistent or therapy refractory CMV infections in allogeneic stem cell transplant patients as an adjuvant to anti-viral therapy.

Characteristics of CMVpp65 CTL Interferon production Cytotoxicity Donor CD4 CD8 BLCL-pp65 BLCL Allo BLCL 1 1% 23% 57% 1% 1% 2 2% 3% 75% 22% 6% 3 6% 1% 31% 7% 7% 4 9% 29% 47% 0% 0% 5 7% 15% 69% 1% 5% 6 24% 3% 10% 1% 6%
Transfection of Bone Marrow Cells In Vitro or In Vivo Prior to Cryopreservation with Manganese Superoxide Dismutase (MnSOD-PL) Protects Frozen Cells from Ionizing Irradiation.
Authors: Epperly Michael W. , Epperly Laura D. , Franicola Darcy , Zhang Xicheng , Smith Tracey , Greenberger Joel S.
Published: April 2020

Overexpression of MnSOD in IL-3 dependent murine hematopoietic progenitor cell line 32D cl 3 protects against apoptosis induced by ionizing irradiation, TNF-α, IL-3 withdrawal or other inducers of cytotoxic stress. Cryopreservaton of bone marrow is routinely used in clinical marrow transplantation with the necessary cytotoxic loss of a fraction of cells from the stress of thawing. To determine if overexpression of MnSOD protected marrow from the stress of thawing and also whether frozen cells resisted ionizing irradiation, 32D cl 3 and subclone 2C6 stably transfected and overexpressing MnSOD were tested. Exponentially growing cells 2C6 cells demonstrate increased irradiation survival with an increased shoulder on the survival curve n = 4.95 ± 0.1 compared to 32D cl 3 n = 2.77 ± 0.1 (p = 0.011), with no significant change in the Do = 2.11 ± 0.08 vs. Do = 2.36 ± 0.10 Gy. Cells from 32D cl 3 and 2C6 were frozen in liquid nitrogen in 50% DMSO and stored at a −140°C for a minimum of 24 hrs and irradiated to doses ranging from 0 to 800 cGy while frozen. The cells were thawed 24 hr later, plated in methylcellulose and colonies of greater than 50 cells counted seven days later. The data was analyzed using linear quadratic and single-hit, multi-target models. Cells from 2C6 were more resistant to thawing with survival of 24.3 ± 0.6% compared to 20.9 ± 0.8% for 32D cl 3 (p = 0.018) and to irradiation as shown by an increase in the Do of 2.036 ± 0.225 compared to 1.455 ± 0.149 Gy for frozen 32D cl 3 cells (p= 0.0287). The frozen 2C6 cells resisted the stress of thawing with an increased plating efficiency compared to the frozen 32D cl 3 cells (121.4 ± 3.2 colonies for 2C6 cells and 104.3 ± 3.9 colonies for 32D cl 3 per 500 cells plated, p=0.0018). To determine whether fresh bone marrow transfected in vivo, or in vitro, then frozen and irradiated, also was protected, C57BL/6NHsd mice were injected with MnSOD-PL (100 ug plasmid DNA in 100 μl). Twenty-four hours later, bone marrow was isolated from MnSOD-PL injected mice as well as control mice (a subpopulation transfected with MnSOD-PL in vitro) and frozen in aliquots of 1 × 106 cells. The cells were irradiated while frozen to doses ranging from 0 to 800 cGy, plated in methylcellulose and colonies of greater than 50 cells counted 7 days later. The colonies were divided between nonadherent and adherent in the methylcellulose. The Do was calculated for the adherent colonies, nonadherent colonies and total colonies. In all three cases the Do for the bone marrow treated with MnSOD-PL in vitro or in vivo was higher than the Do of the control bone marrow. The percent HA-epitope tagged total transfected cells was 22.1 ± 3.7%. The data indicates that MnSOD overexpression protects frozen bone marrow perhaps important to increasing preservation efficiency.
Amplification of Graft vs Malignancy (GVM) Effects by Using Intentionally Mismatched Donor Lymphocytes While Avoiding Graft-vs-Host Disease in Conjunction with Haploidentically Mismatched Stem Cell Allografts in Patients with Hematological Malignances.
Authors: Slavin Shimon , Shapira Michael Y. , Resnick Igor B. , Bitan Menachem , Aker Memet , Samuel Simcha , Morecki Shoshana , Or Reuven
Published: April 2020

Alloreactive donor lymphocytes can induce potent graft vs malignancy (GVM) effects in conjunction with allogenenic stem cell transplantation or when given as donor lymphocyte infusion (DLI) following allogeneic stem cell transplantation. Unfortunately GVM effects are frequently accompanied by graft vs host disease (GVHD) and in many cases, despite severe GVHD, no adequate control of tumor progression can be achieved. In order to improve GVM effects while avoiding GVHD, we have attempted to use natural killer (NK) cells activated with rIL-2 isolated from the blood of haploidentically mismatched donors following engraftment of T-cell depleted donor stem cells. A pilot trial was conducted in patients with hematological malignancies 3 MDS; 2 AML; 1 ALL; 1 biphenotypic leukemia; 5 lymphoma (2 Burkitt’s; 1 anaplastic (ALK) & 1 Hodgkin’s), all resistant to chemotherapy, 4 failing autologous and 1 allogeneic transplant. Eleven received haploidentically mismatched graft. Prevention of rejection was accomplished by activation-induced clonal deletion following infusion of donor buffy coat 24 hours before administration of cytoxan 120–180mg/kg to eliminate host anti-donor T lymphocytes or by using total body irradiation 750cGy following conditioning that was fludarabine based. Positively selected CD34+ cells or CD3 depleted stem cells mobilized with G-CSF were infused on day 0. Based on our murine data, mismatched rIL-2 activated NK cells cause no GVHD. Consequently, 22 procedures were done involving administration of purified NK cells 1–15 months post transplantation. Donor mononuclear cells were activated 4 days at 37°C in 5% CO2 in air with rIL-2 (6,000 IU/ml) and administered on day 4. NK cells were positively selected with anti-CD56, or by negative selection of anti-CD3 using Miltenyi’s immunomagnetic beads. Using this procedure engraftment of mismatched allografts can be accomplished with no or minimal GVHD. Of 11 treated recipients, 5 entered CR. The first patient failing 2 prior transplants from a matched sibling is alive and well 4 years out. Out of 10 patients, 3 died from unrelated causes with no evidence of disease, but no GVHD developed. In conclusion, following engraftment of haploidentically mismatched related stem cell, donor-derived rIL-2 activated NK cells positively selected with anti-CD56 or negatively selected anti-CD3 immunomagnetic beads can be used to induce GVM effects while avoiding GVHD.
A Pilot Trial of Two Dose Levels of Rabbit Antithymocyte Globulin, (rATG), Thymoglobulin, as Part of a Myeloablative-Conditioning for a HLA Identical Matched Related Donor Stem Cell Transplant (SCT) with Cyclosporine (CsA) as Graft Versus Host Disease (GvHD) Prophylaxis.
Authors: Miller Kenneth B. , Chao Nelson J. , Messner Hans A. , Goldstein Steven C. , Reddy Vijay , Vaughan William , Devine Steven , Rowley Scott D. , Waller Ned , Devetten Marcel , Schiller Gary J.
Published: April 2020

To determine the role of rATG to modify the development of acute GvHD we conducted a randomized, prospective, open-label, multi-center pilot study in patients with ALL or AML in first or second remission. CsA was the only agent used for GvHD prophylaxis. Patients (N=22) were randomized (1:1) to receive either 4.5 mg/kg or 8.5 mg/kg of ATG during the preparative regimen. The rATG infusions were completed on Day -1, within 18 hours prior to SCT on Day 0. The low (N=11) and high dose groups (N=11) were comparable for age, diagnosis and remission status. The mean age was 41.4 and 39.6 years in the low and high dose groups respectively. In both groups 6/11 patients had AML and 5/11 had ALL. All patients received a fully ablative preparative regimen from a HLA matched related donor.

Results: Mean time to neutrophil engraftment was 6.5 and 7.3 days for the low and high dose arms, respectively. The incidence of Grade > II acute GvHD was 0/11 for the low dose arm and 3/11 for the high dose arm. Infections were seen in 9/11 patients in both groups. The cumulative number of hospitalization days was 30.4 and 47.6 days (p=n.s.) in the low and high dose groups respectively. The overall survival at 180 days was 63.6 % in the low dose group and 54.5 % in the high dose group (p=n.s.). The disease free survival (DFS) at day 180 was 54.5% in both groups. The treatment related mortality (TRM) was 18.2 % in the low dose and 45.5 % in the high dose (p=n.s.) The relapse rate was 18.2 % in the low dose group and 0% in the high dose group (p=n.s).

Conclusion: This pilot study suggests that rATG may modify the development of serious acute GvHD in patients who received a fully ablative SCT. The low, combined, 3/22, incidence of acute GvHD (13.6%) in patients who received rATG(Thymoglobulin) and post transplant CsA suggests that this combination is an acceptable regiment for GvHD prophylaxis. The lower dose of rATG, 4.5 mg/kg, was as effective and was associated with a lower TRM as compared to the higher, 8.5mg/kg dose. The use of rATG administered during the preparative regimen was well tolerated and may provide an effective alternative to methotrexate when used as part of an ablative conditioning regimen in HLA identical related donor SCT.
Matched Unrelated Donor Stem Cell Transplantation for Patients with Diffuse Large Cell B Cell Lymphoma: A Retrospective Analysis of 118 Patients Registered through the European Group for Blood and Marrow Transplantation (EBMT).
Authors: Avivi Irit , Canals Carme , Taghipour Goli , Niederwieser Dietger , Kanz Lothar , Ehninger Gerhard , Finke Jurgen , Greinix Hildegard T. , Nagler Arnon , Vernant Jean Paul , Maury Sebastien , Bregni Marco , Linkesch Werner , Schwerdtfeger Rainer , Sureda Anna
Published: April 2020

Matched unrelated donor stem cell transplantation (MUD-SCT) may provide a treatment option for patients with diffuse large B cell lymphoma (DLBC) who have failed other conventional therapies and do not have a compatible sibling donor available. We present data of 118 DLBC patients, 69 males and 49 females, aged 18 to 66 years (median 43 years), treated with a MUD-SCT between January 1997 and July 2005 and reported to the EBMT registry. Median time from diagnosis to MUD-SCT was 25 months (range, 3 – 205), and 64% of the cases had failed a previous autologous transplant (ASCT). At allogeneic transplantation, 25% of the patients had chemorefractory disease. Peripheral blood was the source of hematopoietic stem cells in 70% of the cases and reduced intensity conditioning regimens (RIC) were used in 52% of the cases. After a median follow up of 26 months, the estimated 2-year non-relapse mortality (NRM), relapse rate (RR), progression free survival (PFS) and overall survival (OS) for the whole series were 29%, 35%, 36% and 43%, respectively. Grade II–IV acute graft-versus-host-disease developed in 32% of patients. Patients selected for RIC protocols were older (median age of 44 years vs 38 years, p = 0.02) and more heavily pre-treated; 75% had failed a previous autograft compared with 53% in the conventionally treated group (CC) (p = 0.01). Despite these unfavorable factors, the 2-yr NRM for RIC patients was significantly lower than in CC patients: 19% vs 39% (p = 0.03). Unfortunately, this advantage was offset by an increased RR in this group of patients (2-yr RR: 46% vs 24%, p = 0.2), resulting in a very similar PFS and OS for both types of conditioning regimens. The prognostic factor with highest impact on PFS was refractory disease at transplantation (RR = 1.8; 95%CI 1.1 −3.1, p = 0.02). The 2-year PFS for patients transplanted with sensitive disease was 40% irrespective of the conditioning regimen used. In sensitive patients undergoing a RIC transplant, the NRM was significantly lower with respect to CC regimen (14% vs 38%, p = 0.02), resulting in an improved PFS and OS (41% vs 37% and 50% vs 46% respectively). PFS in patients transplanted with refractory disease was generally poor (25% at 2 years). However, CC seemed to provide a better outcome than RIC (2-yr PFS of 35% vs 16%). In conclusion, MUD-SCT constitutes a treatment option for patients with DLBCL failing other conventional treatments, particularly for those patients being allografted in sensitive disease. The high RR observed with reduced intensity protocols does not allow to demonstrate a clear long-term benefit of this approach in this setting.
Development of a Myeloproliferative Disease or a T Cell Lymphoblastic Leukemia in a Murine Bone Marrow Transplant Model of NUP214-ABL1.
Authors: Rocnik Jennifer L. , Cornejo Melanie , Lee Benjamin H. , Okabe Rachel , McDowell Elizabeth , Gozo Maricel , De Keersmaecker Kim , Cools Jan , Gilliland D. Gary
Published: April 2020

Leukemias are often associated with aberrant tyrosine kinase activity that occurs as a result of chromosomal translocations. These mutations are able to confer a proliferative and survival advantage to leukemic cells, as well as cooperate with other mutations that impair cell differentiation, thus leading to the development of leukemia. NUP214-ABL1 is one such recently identified fusion gene that is generated by episomal amplification. The presence of the fusion was recently identified in approximately 6% of patients with T-cell acute lymphoblastic leukemia (T-ALL). By the use of a murine retroviral bone marrow transplantation model we have demonstrated that mice transplanted with NUP214-ABL1 transduced bone marrow cells developed either a myeloproliferative disorder (MPD) with a disease latency of 70 to 118 days or a T cell lymphoblastic leukemia with a disease latency of 115 to 124 days. The myeloproliferative phenotype was characterized by splenomagaly and leukocytosis, and analysis of the histopathology revealed extramedullary hematopoiesis in the liver, lung, kidney and Peyer’s patches, and an increase of peripheral blood neutrophils. Flow cytometry of single cell suspensions from spleen and bone marrow samples of mice with a myeloproliferative phenotype demonstrated an increase of Gr-1+/Mac-1+ cells (approximately 70%). Two of the mice that were transplanted with NUP214-ABL1 transduced bone marrow cells developed T cell lymphomas that were characterized by large thymomas, a phenotype that is consistent with other models of activated tyrosine kinases over long disease latencies. Histopathological analysis of the thymi revealed effacement of normal thymic architecture as well as T cell infiltrate into the surrounding skeletal muscle. In addition, flow cytometric analysis revealed a significant increase in the CD4+/CD8+ T cell population in the thymi of these animals. No disease was observed in secondary transplant recipients following 60 days of observation. In conclusion, these results indicate that NUP214-ABL1 is able to cause either a myeloproliferative disease or a T cell lymphoma over longer latencies in mice, the latter being similar to the phenotype observed in humans with expression of the NUP214-ABL1 fusion. These findings provide a useful model for future experiments to determine if there is a contribution of other mutations together with the NUP214-ABL1 fusion towards the development of a T-ALL phenotype in mice.
Risk of Thromboembolism after Short-Term Interruption of Warfarin.
Authors: Garcia David , Hylek Elaine
Published: April 2020

Background: Significant uncertainty exists for patients who must discontinue warfarin prior to an invasive procedure. In part, the controversy results from the lack of published information about the risk of thromboembolism associated with short-term warfarin interruption. The aim of this observational study was to assess the frequency of thromboembolism and bleeding among a large cohort of patients whose warfarin was temporarily withheld before and after an outpatient invasive procedure.

Methods: A prospective cohort of patients taking warfarin was assembled from 101 sites with personnel designated as warfarin managers across the United States during the period August 2000 to December 2001; 98 sites were community-based physician office practices. All episodes of warfarin interruption among the 6,761 patients in the cohort were identified by investigator review of provider-generated progress notes. Surgical procedures (including cardiovascular interventions) that required hospitalization and cases in which follow-up INR values were not available for at least 30 days post-procedure were excluded. The primary outcomes (30 day follow-up) were

thromboembolism and clinically significant hemorrhage.

For each patient, the following data were also recorded: type of procedure, number of days warfarin was withheld, presence or absence of bridging therapy (e.g. low molecular weight heparin [LMWH]).

Results: 1,024 unique patients (1,293 episodes of warfarin interruption) were included in our analysis. The mean age was 71.9 years; 43% were female. The most common procedures were colonoscopy (n=324), oral surgery (n=323), and ophthalmic surgery (116). Other common reasons for withholding warfarin were: epidural injection, prostate biopsy, breast biopsy, and dermatologic procedures. Perioperative heparin or LMWH was used in only 8.3% of cases. The duration of warfarin interruption was variable; however, more than 80% of patients had warfarin withheld for 5 days or fewer. Seven patients (0.7%) suffered post-procedure thromboembolism within 30 days (4 arterial, 3 venous). None of the 7 patients who experienced thromboembolism received peri-procedural bridging therapy; 3 of these thromboembolic events were associated with warfarin interruption ≥ 7 days. Twenty-three patients (2.3%) suffered clinically significant hemorrhage - of these, 14 had received peri-procedural heparin or LMWH.

Conclusion: For many chronically anticoagulated patients who need to undergo a minor, outpatient intervention, a brief (5 or fewer days) peri-procedural interruption of warfarin is associated with a low risk of thromboembolism. The risk for clinically significant bleeding, even among outpatients undergoing minor procedures, should be considered prior to the administration of bridging therapy.
Pure Heterozygous and Mixed JAK2 V617F Genotypes Result in Distinct Mechanisms of Erythrocytosis in Polycythemia Vera.
Authors: Dupont Sabrina , Masse Aline , James Chloe , Casadevall Nicole , Vainchenker William , Delhommeau Francois
Published: April 2020

The JAK2 V617F mutation is present in most patients with polycythemia vera (PV) and half with essential thrombocythemia (ET). Using real-time quantitative PCR, we analyzed the levels of JAK2 V617F in granulocytes and/or bone marrow mononuclear cells from 159 PV and 149 ET patients. High JAK2 V617F levels were correlated with higher leukocyte, granulocyte, hemoglobin values and higher endogenous erythroid colony formation. Thus, the phenotype of PV and ET may be closely linked to the JAK2 V617F level, which may reflect the clonal genotypic pattern of hematopoietic progenitor cells. It is thought that the occurrence of the mitotic recombination, which generates homozygous JAK2 V671F clones, is a key molecular event for the onset of PV. In this work, we aimed to study the consequences of the clonal JAK2 V617F genotype on the amplification properties and erythropoietin (EPO) hypersensitivity of PV (n=14) and ET (n=6) progenitors.

Analysis of clonal genotypic patterns shows that ET patients harbor a mix of heterozygous and normal progenitors. Interestingly, we distinguish pure heterozygous PV profiles (3/14 patients) with no homozygous progenitors from homozygous PV profiles (11/14 patients) with normal, heterozygous and homozygous progenitors. Similar low frequencies of mutated immature progenitors, comprising long-term culture-initiating cells and lympho-myeloid progenitors, are found in ET and PV. In contrast, PV patients with pure heterozygous PV profiles have striking higher proportions (>90%) of mutated committed progenitors than other PV and ET patients. This result suggests a selective amplification of heterozygous cells in the early phases of hematopoiesis. Furthermore, by using increasing concentrations of EPO, homozygous mutated erythroid progenitors are demonstrated to be more sensitive to EPO than heterozygous ones, a majority of the former (69,5%) being EPO independent. Moreover, we demonstrate a two to three fold increase in in vitro amplification of ET and PV progenitor cells when compared to normal ones in serum free liquid culture containing IL3, Stem Cell Factor, Dexamethasone and 1 IU/mL EPO. In addition, the quantification of the mutated allele in immature CD34+CD38- cells, in CD34+CD38+ committed progenitor cells, in mature erythroblasts (GPA+) and in granulocytes shows a marked in vivo selective advantage of mutated cells in late stages of hematopoiesis.

These results suggest that in PV, erythrocytosis results from two distinct mechanisms: a terminal erythroid amplification advantage triggered by homozygosity or a two-step process including the upstream amplification of heterozygous cells that may involve additional molecular event(s).
Prostaglandin E2 Is a Potent Regulator of Vertebrate Hematopoietic Stem Cell Homeostasis.
Authors: North Trista E. , Goessling Wolfram , Kopani Kamden R. , Lengerke Claudia , Walkley Carl R. , Lord Allegra M. , Jang Il-ho , Grosser Tilo , FitzGerald Garret A. , Daley George Q. , Orkin Stuart H. , Zon Leonard I.
Published: April 2020

Hematopoietic stem cells (HSCs) give rise to each of the blood lineages found in the adult vertebrate. Definitive HSCs are derived during embryogenesis in the aorta-gonad-mesonephros (AGM) region and subsequently colonize the niche in fetal and adult hematopoietic organs. To identify regulators of this process, a library of biologically active compounds was screened for effects on zebrafish HSC development. Embryos were exposed to individual chemicals (2498) from the 5-somite stage until 36 hpf, then processed by whole-mount in situ hybridization for the expression of HSC markers cmyb and runx1. Exposure to 47 of the compounds reduced the number of runx1+cmyb+ HSCs, while 35 increased this cell population. Several chemicals isolated in the screen were known to affect the prostaglandin (PG) synthesis pathway. Prostaglandin (PG) precursors, mead acid and linoleic acid, expanded HSCs, while fenbufen and celecoxib, inhibitors of cyclooxygenase (cox) activity, reduced HSC number. An examination of blood and vessel specific molecular markers demonstrated that angiogenesis was not altered significantly in treated embryos, illustrating the specific effect on HSCs. Exposure to exogenous PGE2 modestly increased HSCs, while use of a long-acting version of PGE2, 16,16-dimethylPGE2 (dmPGE2), markedly enhanced HSC number. Incubation of zebrafish embryos with known cox inhibitors confirmed the observation that inhibition of PG synthesis reduces HSCs. Injection of morpholino oligonucleotides designed against cox1, cox2 and PGE synthase inhibited the formation of HSCs in a dose dependent fashion, verifying that signaling through PGE2 is regulating AGM HSC number. Using CD41:GFP;LMO2:DsRed bigenic zebrafish, the modulation of HSCs following exposure to dmPGE2 or indomethacin was documented in vivo by confocal microscopy. Exposure to dmPGE2 following irradiation of adult fish induced HSC gene expression and enhanced hematopoietic recovery, indicating that PGE2 also functions in regulating adult marrow HSC homeostasis. Incubation of murine embryonic stem cells with PGE2 during embryoid body differentiation resulted in a dose-dependent increase of hematopoietic colonies in methylcellulose, while indomethacin exposure inhibited colony formation, demonstrating that the function of PGE2 in HSC regulation is conserved in mammals. Furthermore, ex vivo treatment of murine bone marrow with dmPGE2 resulted in a 2-fold increase in CFU-S number and enhanced the frequency of multi-lineage engrafting HSCs more than 3-fold in a limiting dilution competitive repopulation assay. The identification of the prostaglandin synthesis pathway as a potent regulator of vertebrate hematopoietic stem cell homeostasis has major implications for new treatments of diseases such as anemia and leukemia.