bluuebunny/crossref_metadata_2025 huggingface dataset:
Human Neutrophil Elastase Stimulating CD4+ and CD8+ T Cells Is a Potential Protein Vaccine for Leukemia Patients with Diverse HLA Types.
Authors: Le Quan , Melenhorst J. Joseph , Hensel Nancy , Eniafe Rhoda , Barrett A. John
Published: April 2020

The detection of naturally occurring T cells responding to proteins overexpressed in myeloid leukemia suggests that vaccines could boost the immune response to leukemia and achieve disease control. Human neutrophil elastase (HNE) is a myeloid-restricted protein highly expressed in myeloid leukemia cells. HNE protein can induce both CD4+ and CD8+ T cell responses in healthy individuals including HLA-A*0201 negative individuals. To develop HNE protein vaccine, and to identify major histocompatibility complex (MHC) class II restricted or HLA-A*0201 negative MHC class I restricted epitopes in HNE, we stimulated peripherial blood mononuclear cells (PBMC) from 6 myeloid leukemia pre-transplant patients with purified full length HNE protein. HNE protein induced a response in CD4+ and CD8+ T cells from 4 patients with acute myeloid leukemia (AML), as measured by IFN-γ protein expression in flow cytometric assay. However, no response was detected in CD4+ and CD8+ T cells from 2 pre-transplant patients with chronic myeloid leukemia (CML). Responses of CD4+ and CD8+ T cells to HNE protein stimulation were enhanced by HNE expressing antigen-presenting T cells (T-APC), as measured by flow cytometric assay. HNE protein induced significant IFN-γ expression in CD4+ and CD8+ T cells at 540X and 10X above background in an (HLA-A11, 31) AML patient (patient #1 in the figure), and at 6X and 3.5X above background in an (HLA-A*0205, 03*) AML patient (patient #2 in the figure), as measured by quantitative Real-Time RT-PCR assay (QRT-PCR). HNE protein also induced significant IL-2 production in CD4+ and CD8+ T cells at 7.7X and 6.1X above background in patient #1. Finally, we showed by QRT-PCR that the T cell responses to HNE protein stimulation were associated with the levels of HNE gene transcripts in the PBMC. Thus we found that full-length HNE protein stimulated both CD4+ and CD8+ T cell responses in HLA-A*0201 negative patients with AML. These results provide the rationale for using the full-length HNE protein to vaccinate patients with myeloid leukemias pre- or post-stem cell transplantation to improve graft-versus-leukemia effects.

Figure Figure
Determining the Mechanism of Transformation of Follicular Lymphoma into Diffuse Large B Cell Lymphoma.
Authors: Carlotti Emanuela , Wrench David , Iqbal Sameena , O’Shea Derville , Davies Andrew , Virappane Priya , Hart Jason , Lai Raymond , Gribben John G. , Lister Andrew T. , Fitzgibbon Jude
Published: April 2020

The molecular mechanisms involved in histological transformation of follicular lymphoma (FL) to diffuse large B-cell lymphoma (DLBCL) are largely unknown. Here we investigated the clonal relationship in FL and DLBCL in 17 cases of paired FL/DLBCL samples and also in a bone marrow transplant (BMT) model. The aim of this study was to determine the cell of origin of the transformed DLBCL (t-DLBCL) and whether this cell always arises as a clonal evolution from the major FL clone. The immunoglobulin variable heavy chain (IgVH) was amplified by PCR using family specific primers, the major clone identified by homo/heteroduplex and genealogical trees generated. As expected 70% of FL/DLBCL cases used VH3, 24% VH4 and 6% VH5. The median degree of somatic hypermutation (SHM) was 15% (range 4 to 43%). In 3/17 cases the pattern of SHM was identical between FL and DLBCL. In 6/17 cases a clear pattern of direct evolution was evident. Surprisingly for 8/17 cases the pattern of SHM was compatible with the FL and DLBCL arising from a precursor cell rather than direct evolution. Clone specific primers and probes were designed to investigate whether there was evidence of the t-DLBCL in the earlier biopsy. Quantification by RQ-PCR demonstrated that in most cases the transformed clonotype was already present at levels between 10-4 and 10-2. We further examined an interesting case in which both the father (donor) and the son (recipient) developed FL and t-DLBCL 3 and 10 years after transplantation respectively. Sequencing confirmed the presence of an identical t(14;18) translocation in donor and recipient and that both cases used IGHV3-48*03. Analysis of the genealogical trees of SHM in these cases was also consistent with the evolution of t-DLBCL from a common precursor cell rather than direct evolution. These data are consistent with two distinct patterns of transformation of FL to t-DLBCL. About 50% of our cases had evidence of direct evolution while in the remaining 50% the pattern is more suggestive of the FL and t-DLBCL arising from a common precursor cell. The long latency period after BMT is in agreement with the requirement for additional genetic events within the t(14;18) bearing post germinal centre precursor lymphoma cell for the development of these diseases. The finding of the t-DLBCL clonotype in the FL sample before transformation suggests that the transforming event may occur in a minor subclone of the disease or earlier in a lymphoma precursor cell. Ongoing experiments seek to investigate the genetic events arising in these cells that result in transformation.
Apoptosis Inducing Factor Acts as a Survival Factor in Acute Myelogenous Leukemia through Mitochondrial Complex 1.
Authors: Wang Rui-yu , Samudio Ismael J. , McQueen Teresa , Schober Wendy D. , Ruvolo Vivian R. , Kornblau Steven , Konopleva Marina , Andreeff Michael
Published: April 2020

Apoptosis inducing factor (AIF) is a mitochondrial flavoprotein that is released into the cytoplasm to form an active DNase and then translocates into the nucleus to induce DNA fragmentation when cells undergo apoptosis. AIF also acts as an essential NADPH oxidase, a function which is distinct from its proapoptotic role. We have shown previously that AIF expression level increased with increasing blast counts in primary acute myelogenous leukemia (AML) patient samples. Here we report that AIF expression levels are increased in primary CD34+ AML cells compared with CD34− cells. To investigate the role of AIF in primary AML, bone marrow or peripheral blood samples from AML patients (n=8) with CD34+ or CD34− cell populations were examined for changes in reactive oxygen species (ROS), mitochondrial membrane potential (ΔψM), apoptosis and chemo-sensitivity in the CD34+ and CD34− compartments after treatment with the mitochondrial complex 1 inhibitor rotenone. Similar experiments were performed following knockdown of AIF with lentiviral shRNA directed against AIF in K562 cells.

Results demonstrate:

significantly increased apoptosis rate (39.2% Annexin V+ cells in CD34+ vs. 14.1% in CD34− cells following 2 uM rotenone, p=0.05), and decreased ΔψM in CD34+ cells (44.5% ΔψM cells in CD34+ vs. 78.2% in CD34− cells, p=0.01). MCL-1 expression levels were significantly decreased in CD34+ but not in CD34− cells (p=0.007). The apoptotic effect of rotenone in CD34+ cells was potentiated by AraC (1uM). Profound inhibition of O2 consumption in AIF knockdown K562 cells (−5.2±0.3) compared with K562-neo (neo transfected K562, −13.1±0.3) cells (p=0.001).

Rotenone treated AIF knockdown K562 cells and K562-neo cells revealed that decreased AIF expression led to higher ΔψM and higher superoxide generation (p=0.0002). In contrast, total mitochondrial mass (NAO) decreased slightly in K562KD cells. Data suggested that

AIF contributes to leukemia cell survival by modulating mitochondrial metabolism, independent of its proapoptotic activity, which is consistent with recent reports demonstrating its importance in mitochondrial complex 1 activity. Decreased AIF levels are associated with increased generation of superoxide anion, suggesting that AIF critically regulates mitochondrial function in AML cells. Rotenone selectively decreases AIF and induces apoptosis in CD34+ cells in AML. AIF functions as a survival factor in AML strategies targeting AIF expression or function may be beneficial in the treatment of myeloid leukemia.
The RARa-PLZF Oncogenic Protein Inhibits C/EBPa Function in Myeloid Cells.
Authors: Tremblay Mathieu , Girard Nathalie , Haman Andre , Grondin Benoit , Bouchard Nathalie , Labrecque Jean , Chen Bing , Chen Zhu , Chen Sai-Juan , Hoang Trang
Published: April 2020

In acute promyelocytic leukemia (APL), the variant t(15;17) translocation is responsive to differentiation therapy with retinoic acid (RA) while the t(11;17) APL is a more aggressive disease with poor prognosis. The latter produces two fusion proteins, PLZF-RARa and RARa-PLZF, and both proteins are required for leukemogenesis. To define the role of RARa-PLZF, we ectopically expressed the fusion gene in 32D cells and in primary bone marrow cells. First, our results show that RARa-PLZF inhibits myeloid gene expression, specifically CEBPa targets, which fulfill important function in cell survival and differentiation along the granulocytic lineage. Second, we found that repression by RARa-PLZF is dependent on the binding of C/EBPa to its cognate sequence in the promoter of CEBPa target gene, GCSFR. Third, we confirmed by chromatin immuprecipitation that RARa-PLZF associate with C/EBPa on DNA. Fourth, we showed that as PLZF, RARa-PLZF interact directly with HDAC1 and that this interaction causes a deacetylation of histone H3 at the promoter. This inhibition is reversed by treatment with histone deacetylase inhibitor (TSA) both in vitro and in vivo. Thus, this repression is dependent on direct interaction of RP with C/EBPa and recruitment of HDAC1, causing histone deacetylation at C/EBPa target loci. Finally, our data indicate that C/EBPa activity is severely impaired in leukemic cells from patients with t(11;17) APL, as compared to the t(15;17) APL, which is more amenable to therapy. In summary, our study indicates that the oncogene RARa-PLZF inhibits C/EBPa function through direct protein-protein interaction, and thus contributes to leukemogenesis in t(11;17) APL.
The Presence of Central Nervous System Disease at Diagnosis in Pediatric Acute Myeloid Leukemia Does Not Affect Outcome.
Authors: Johnston Donna L. , Alonzo Todd A. , Gerbing Robert B. , Lange Beverly J. , Woods William G.
Published: April 2020

Introduction: The presence of disease in the central nervous system (CNS) in pediatric acute myeloid leukemia (AML) is often thought to confer a worse prognosis. This study examined the outcome of children with AML who had CNS disease at diagnosis.

Methods: Patients enrolled on Children’s Oncology Group protocols 2891 (N=836)and 2961 (N=901) being treated for de novo AML were classified for the presence of CNS disease at diagnosis as: CNS1 (less than 5 WBC in the CSF with no blasts), CNS2 (less than 5 WBC in the CSF with blasts) or CNS3 (5 or more WBC in the CSF with blasts). CNS disease at diagnosis was then analysed for survival, patient characteristics and outcome using univariate analysis. Patients on these protocols were treated with intrathecal chemotherapy and not radiation therapy for their CNS disease.

Results: For both AML protocols, the overall survival and event free survival were highest in patients with CNS2 disease but this was not significantly different from the survival of the CNS1 and CNS3 patients.

Overall survival from 2891 study entry for all de novo patients Overall survival from 2891 study entry for all de novo patients Overall survival from 2961 study entry for all de novo patients Overall survival from 2961 study entry for all de novo patients

Patients with CNS 2 and 3 disease were of significantly lower median age than CNS1 patients (p=0.001 in 2891 and p=0.005 in 2961). There was a significantly higher WBC count at diagnosis (p=0.001 in both studies), presence of hepatomegaly (p=0.001 in both studies), presence of splenomegaly (p=0.003 in 2891 and p=0.001 in 2961), FAB M2 morphology (p=0.001 in both studies) and FAB M4 morphology (p=0.001 in both studies) in patients with CNS2 and 3 disease compared to CNS 1 patients. Also, CNS2 and 3 patients had a significantly higher incidence of abnormal chromosome 16 (p=0.002 in 2891 and p=0.001 in 2961). In protocol 2891 significantly more CNS2 and 3 patients had hyperdiploid cytogenetics compared to CNS1 patients (p=0.016), and in protocol 2961 there were significantly more patients with t(8;21) in the CNS1 group (p=0.007). In terms of overall outcome, there was a significantly higher incidence of isolated CNS relapse in patients with CNS3 disease at diagnosis in the 2891 protocol (p=0.001), and in patients with both CNS2 and 3 disease at diagnosis in the 2961 protocol (p=0.001).

Conclusion: Patients with CNS disease at diagnosis have similar survival to those without CNS disease, although they have an increased incidence of isolated CNS relapse. More aggressive CNS directed therapy may be warranted in this patient population.
High Frequency of 1p36.32 Deletion or Loss of Heterozygosity in Follicular Lymphoma (FL).
Authors: Cheung K.-John , Telenius Adele , Lai Betty , Johnson Nathalie , Relander Thomas , Steidl Christian , Baross Agnes , Qian Hong , Schein Jacquie , Marra Marco , Connors Joseph M. , Gascoyne Randy D. , Horsman Douglas E.
Published: April 2020

Background: The initial genetic event in ∼85% of follicular lymphomas (FL), the most common B-cell lymphoma in North America, is the t(14;18)(q32;q21) resulting in over-expression of the anti-apoptotic protein Bcl-2. The secondary events associated with disease progression are not well understood. Alterations affecting the p arm of chromosome 1 are evident by standard karyotype analysis in ∼20% of FL. We have further examined the relationship between 1p deletion and FL using high resolution genomic analyses.

Methods: The prevalence of 1p alterations was investigated in 139 cases of indolent and transformed FL using whole genome tiling path BAC array Comparative Genomic Hybridization (array CGH). Array-based single nucleotide polymorphism analysis was performed on a subset of cases using Affymetrix 500K SNP arrays.

Results: Array CGH identified a minimum region of deletion spanning ∼0.5MB within 1p36.32 in 51 cases (37%). In 38 cases (27%) this loss was exhibited in the transformed sample but not the pre-transformation sample. The majority of cases displayed heterozygous deletion, while two cases showed homozygous deletion. The mechanisms of loss included simple deletions, unbalanced translocations with various partner chromosomes and eleven cases with an unbalanced t(1;1)(p36;q12). The Affymetrix 500 SNP array analyses showed copy neutral loss of heterozygosity or acquired uniparental disomy (aUPD) in three of ten cases that were negative for loss by aCGH. Contained within the 1p36.32 minimally deleted region are only a few candidate genes including tumor necrosis factor receptor superfamily 14 (TNFRS14), which has been implicated in growth inhibition of HT-29 human colon adenocarcinoma cells and induction of Fas-mediated apoptosis in non-Hodgkin’s lymphoma.

Conclusions: Our data indicate that loss of heterozygosity at 1p36.32 through deletion or aUPD constitutes the most common secondary cytogenetic event in FL. LOH at 1p36 may represent an important step in the progression of indolent to transformed FL. Further studies have been initiated to investigate other possible gene inactivation events such as methylation and mutation.
Preliminary Results of a Prospective Multicenter Phase III Trial Comparing High-Dose and Standard-Dose Daunorubicin for Induction of Complete Remission (CR) in Acute Myeloid Leukemia (AML).
Authors: Lee Je-Hwan , Joo Young-Don , Park Jae-Hoo , Ryoo Hun Mo , Hyun Myung Soo , Zang Dae Young , Ahn Jin Seok , Lee Junglim , Lee Kyeong Won , Choi Seong-Jun , Lee Jung-Hee , Seol Miee , Kim Keun-Hee , Lee Won-Sik , Min Young Joo , Kim Hawk , Bae Sung-Hwa , Kim Min Kyung , Kim Hyo Jung , Lee Kyoo-Hyung
Published: April 2020

The effectiveness of anthracycline dose intensification for induction of CR in AML has not been studied in a randomized fashion. We conducted a prospective randomized trial to compare the therapeutic efficacy and toxicity of two different doses of daunorubicin in combination with cytarabine in AML. This study began on August 2001 and 293 adult patients (younger than 60 years) with newly diagnosed AML except M3 have been enrolled. Fourteen patients were removed from the study and the remaining 279 patients were analyzed. After random assignments, 135 patients received standard-dose daunorubicin (SD-DN, 45 mg/m2/d × 3 d) and 144 patients received high-dose daunorubicin (HD-DN, 90 mg/m2/d × 3 d) in addition to cytarabine (200 mg/m2/d × 7 d) for induction of CR. Patients with persistent leukemia received the second attempt of induction chemotherapy, consisting of standard-dose daunorubicin (2 d) plus cytarabine (5 d). Patients who attained CR received 4 cycles of high-dose cytarabine (3 g/m2 × 6 doses) and 2 cycles of daunorubicin (1 d) plus cytarabine (5 d). For patients in intermediate- or high-risk cytogenetic groups, allogeneic hematopoietic cell transplantation was performed if there was a suitable donor. CR was induced in 98 of 135 patients (72.6%) in SD-DN arm and 119 of 144 (82.6%) in HD-DN arm (P = 0.044). The impact of daunorubicin dose intensification on the CR rates were different by cytogenetic risk group: the CR rates for SD-DN vs. HD-DN arm were 24/25 (96.0%) vs. 35/36 (97.2%) for good-risk group, 63/88 (71.6%) vs. 64/83 (77.1%) for intermediate-risk group, and 10/20 (50.0%) vs. 19/24 (79.2%) for poor-risk group. With a median follow-up of 596 days for surviving patients, the 4-year probabilities of overall survival, disease-free survival, and relapse-free survival were similar between SD-DN and HD-DN arm. Two different doses of daunorubicin (SD-DN vs. HD-DN) showed similar toxicity profiles regarding recovery times from myelosuppression, transfusion requirements, severe toxicities (grades III to IV) classified by NCI-CTC ver 2.0 including cardiac toxicities, and duration of antibiotics administration. In conclusion, high-dose daunorubicin showed higher CR rates in AML patients of intermediate- and poor- cytogenetic risk groups without increase of toxicities compared to standard-dose daunorubicin.
An Open Label, Dose Escalation Study of AVE9633 Administered as a Single Agent by Intravenous (IV) Infusion Weekly for 2 Weeks in 4-Week Cycle to Patients with Relapsed or Refractory CD33-Positive Acute Myeloid Leukemia (AML).
Authors: Legrand Ollivier , Vidriales Maria B. , Thomas Xavier , Dumontet Charles , Vekhoff Anne , Morariu-Zamfir Rodica , Lambert John , San Miguel Jesus F. , Marie Jean-Pierre
Published: April 2020

AVE9633/huMy9-6-DM4 is an immunoconjugate composed of a humanized monoclonal IgG1 antibody, huMy9-6, which specifically targets the CD33 antigen, conjugated through a disulfide link to the maytansine derivative DM4, a potent tubulin inhibitor. The CD33 antigen is expressed on the surface of myeloid cells. After the conjugate is bound to the CD33 antigen it is internalized and the cytotoxic is released within the target cell. We report preliminary results of the ongoing phase I dose escalation study of AVE9633 in patients with refractory/relapsed CD33+ AML. The study regimen consists of AVE9633 IV infusion on Day 1 and Day 8 of a 28-day cycle. To date, dose levels of 30 (n=1), 50 (n=3), 75 (n=3), 105 (n=3) and 130 (n=3) mg/m2 on Day 1 and Day 8 have been investigated. Patients received 1 (n=2), 2 (n=5), 3 (n=2) and 4 (n=4) cycles of AVE9633. No dose-limiting toxicity was noted so far. Nine patients experienced mild to moderate infusion reactions, mostly on Day1 of Cycle1. One patient at 130 mg/m2 presented grade 3 ALT elevation of 3 days duration. Free DM4, measured by LC/MS/MS was detectable at 105 and 130 mg/m2 in the range of 5 to 10 ng/mL. AVE9633/huMy9-6-DM4 exposure (measuring, by ELISA method, all antibodies containing at least one molecule of DM4) increased proportionally with the administered dose. Using Flow Cytometry Assay, saturation and down regulation of CD33 on peripheral and marrow blasts were observed from the dose of 50 mg/m2. There was one CRp in a 68-year old woman with refractory AML, achieved after 4 cycles of AVE9633 given at 105 mg/m2 × 2, with a 4-month continuing CRp as of 7/07 (she is receiving AVE9633 105 mg/m2 once monthly). One PR occurred in a 81-year old woman with refractory AML after one cycle at 130 mg/m2 × 2, persistent after the second cycle (third cycle is ongoing). Two patients had >50% decrease in bone marrow blasts, at 105 mg/m2 × 2 (from 25% to 8% after two cycles) and at 130 mg/m2 × 2 (from 35% to 9% after one cycle). One additional patient at 75 mg/m2 × 2 presented clearance of peripheral blasts by Day 10 of Cycle1. Mean DM4/IgG on the surface of peripheral blasts (available for one patient at 130 mg/m2) progressively decreased after the infusion, from 3.09 on day 1 to 0.28 on Day 8, and then increased to 2.64 after the second infusion of AVE9633 on Day 8. These preliminary results show that AVE9633 is well tolerated, with manageable safety profile, allowing outpatient treatment. Evidence of anti-leukemia activity was observed in 5 patients. The study is continuing with the evaluation of 150 mg/m2 × 2. Evaluation of closer drug administrations which might enhance efficacy appears warranted.
Safety and Efficacy of Alfimeprase in Subjects with Occluded Central Venous Access Devices: The SONOMA-2 Study.
Authors: Moll Stephan , Gabrail Nashat , Buchanan Jim , Hirmand Mohammad , Levy Michael , Zitnik Ralph
Published: April 2020

Introduction: The infusion of chemotherapy and blood products is facilitated by central venous access devices (CVAD), and the occurrence of thrombotic CVAD occlusion results in delays in these crucial therapies. Alfimeprase is a direct-acting thrombolytic agent with the potential to rapidly re-establish catheter function.

Methods: SONOMA-2 was a multicenter, multinational, randomized, double blind, placebo-controlled phase 3 study evaluating the efficacy and safety of alfimeprase in subjects with occluded CVADs. Subjects were randomized in a 2:1 ratio to receive either intra-luminal alfimeprase (A) at a dose of 3.0 mg or placebo (P). Study drug was delivered in a total volume of 2 mL. If re-establishment of catheter function was not established within 30 minutes, a second dose of the same study drug was instilled and allowed to dwell for an additional 30 minutes. Re-establishment of catheter function was assessed by an attempt to withdraw 3 mL of blood and infuse 5 mL of saline. The primary efficacy endpoint was re-establishment of a functional CVAD at 15 minutes post first infusion. The primary efficacy analysis prespecified a significance level of ≤ 0.00125.

Results: A total of 303 patients (201 (A), 102 (P)) were randomized into the study. Treatment groups were well balanced at baseline with respect to overall demographics, catheter insertion site, and median days since last failed infusion or blood draw. The primary efficacy endpoint, re-establishment of a functional CVAD at 15 minutes post first infusion, was achieved in 34.3% (A) versus 21.6% (P) of subjects (p = 0.022). The cumulative proportion of subjects with re-establishment of a functional CVAD at 30 minutes post first or second infusions was 52.7% (A) versus 30.4% (P) (p = 0.0002). Rates of adverse events (65.8% (A), 59.4% (P)), serious adverse events (15.5% (A), 16.8% (P)), and hemorrhagic adverse events (3.6% (A), 10.9% (P)) were comparable between treatment groups. No intracranial hemorrhages were observed.

Conclusions: Alfimeprase restored CVAD function in a numerically greater fraction of subjects than did placebo; however, the study did not achieve statistical significance for the primary endpoint at the prespecified p-value of ≤ 0.00125 necessary for demonstrating efficacy with a single pivotal study. Alfimeprase was well tolerated. Further clinical studies are underway that evaluate a higher and more concentrated alfimeprase dose which preclinical studies predict will result in greater efficacy. Alfimeprase remains a promising potential candidate for use in patients with CVAD occlusion.
Time of Relapse after Initial Therapy Significantly Adds to the Prognostic Value of the IPI-R in Patients with Relapsed DLBCL Undergoing Autologous HSC Transplantation.
Authors: Costa Luciano J. , Micallef Ivana N. , Inwards David J. , Johnston Patrick B. , Porrata Luis F. , Ansell Stephen M.
Published: April 2020

Background: High-dose chemotherapy and hematopoietic stem cell (HSC) transplantation is curative in about 40% of patients with relapsed chemosensitive diffuse large B cell lymphoma (DLBCL). The international prognostic index obtained at the time of relapse (IPI-R) and the time from initial treatment to relapse (TTR) have been suggested in different series to be predictors of outcome after HSC transplantation. We explored the concomitant effect of IPI-R and TTR on therapeutic outcome in a modern and uniform cohort of patients with relapsed chemosensitive DLBC undergoing high-dose chemotherapy and peripheral HSC transplantation.

Methods: Retrospective analysis of 80 consecutive patients treated at Mayo Clinic, Rochester, MN between 2001 and 2006 who received a peripheral HSC transplant (except for 3 patients who receive bone marrow) after being conditioned with BCNU, etoposide, cytarabine and melphalan (BEAM) for relapsed chemosensitive DLBCL.

Results: Median follow-up of survivors was 31.4 months, Median age at the time of transplant was 62 (interquartile range 53–65; range 26–77), 65% were males; median number of prior chemotherapy regimens was 2. All patients had received at least one previous anthracycline-based regimen, 78% had been previously exposed to rituximab and all achieved at least CRu after the initial treatment. The median time from diagnosis to relapse was 19 months (interquartile range 10–43), 25% of patients had a high or high-intermediate IPI-R. The median survival from the time of transplant for patients with TTR>18 months v. ≤ 18 months was not reached and 50 months respectively (P=0.005). Median survival for patients with high or high-intermediate IPI-R at the time of relapse (IPI score ≥3) was 23.3 months and not reached for patients with low or low-intermediate IPI (P=0.01). These two factors were independent in multivariate analysis with RR for death of 1.8 (1.2–2.9; P=0.004) for TTR ≤ 18 months and 1.7 (1.1–2.5; P=0.017) for high or high-intermediate IPI and therefore combined in a prognostic system were patients with none (n=32), one (n=39) or two (n=9) of these prognostic factors had median disease free survival not reached v. 50 months v. 3.3 months (P=0.001) and overall survival not reached v. 50 months v. 5 months (P=0.0001) (Figure).

Conclusion: TTR and IPI-R provide an easy and powerful prognostic system in patients with DLBCL undergoing modern high-dose chemotherapy with HSC support.

Figure Figure
Multicenter Phase II Trial of 90Y-Ibritumomab Tiuxetan with High-Dose Chemotherapy (Busulfan/Cyclophosphamide/Etoposide) Followed by Autologous Stem Cell Transplantation in Relapsed, Refractoried, or High-Risk B-Cell NHL.
Authors: Kang Byung Woog , Jo Jae-Cheol , Kim Shin , Jang Geundoo , Lee Sung Sook , Sym Sun Jin , Ryu Jin-Sook , Kim Won Seog , Yoon Sung Soo , Suh Cheolwon
Published: April 2020

The need of new effective regimen for high-dose chemotherapy followed by autologous stem cell transplantation (ASCT) in aggressive B-cell non-Hodgkin’s lymphoma (NHL) patients and promising results observed so far in trials with 90Y-Ibritumomab tiuxetan containing regimens in ASCT strongly warrants the investigation of 90Y-Ibritumomab tiuxetan combined busulfan/cyclophosphamide/etoposide (Z-BuCyE) high-dose chemotherapy with ASCT for relapsed, refractoried, or high-risk B-cell NHL. We evaluated efficacy and safety of the combination of Z-BuCyE and ASCT in patients with relapsed, refractoried, or high-risk B-cell NHL. Treatment consisted of two doses of Rituximab (250 mg/m2, IV, day -21, -14) and a single dose of 90Y-Ibritumomab (0.4 mCi/kg, IV, day -14). All patients received conditioning regimen: busulfan (3.2 mg/kg, IV, day -7, -6, -5), etoposide (200 mg/m2, IV, day -5, -4), and cytoxan (50 mg/kg, IV, day -3, -2) followed by ASCT (day 0). Thirteen patients were entered the trial. The median age was 46.1 years (range: 25–60), and 6 (46%) patients were male. Histology was diffuse large B-cell (n=10), follicular (n=1), Burkitt (n=1), and mantle cell lymphoma (n=1). The objective overall response rate (ORR) was 76.9% (10/13): continued CR, 38.5% (5/13); induced CR, 23.1% (3/13); continued or induced PR, 15.4% (2/13). Three patients (23.1%) had a PD after transplantation and two of these patients died of progression. Median follow-up duration was 6.0 months. Median progression-free survival (PFS) and median overall survival (OS) has not yet been reached. Toxicity was principally non-hematologic. Grade 2 toxicity included mucositis (53.8%), nausea (61.5%), vomiting (15.4%), diarrhea (23.1%), and elevation of liver enzyme (7.7%). Grade 3 toxicity included mucositis (15.4%), nausea (23.1%), and diarrhea (23.1%). There was no grade 4 toxicity. Infection occurred in ten patients, bleeding in one patient, and there was no treatment related mortality. This preliminary analysis shows that the combination of Z-BuCyE and ASCT has excellent efficacy and is well-tolerated treatments for relapsed, refractoried or high-risk B-cell NHL. This study will be continued till 20 patients enrollment.
Retroviral Vector-Producing Mesenchymal Stem Cells for Tumor Tracking and Therapeutic Gene Amplification in Suicide Cancer Gene Therapy.
Authors: Uchibori Ryosuke , Okada Takashi , Ito Takayuki , Urabe Masashi , Mizukami Hiroaki , Kume Akihiro , Ozawa Keiya
Published: April 2020

Mesenchymal stem cells (MSCs) are known to have a tendency to accumulate at the site of tumors, and therefore can be utilized as a platform for targeted delivery of anti-cancer agents. The MSC-based targeted cancer gene therapy can enhance the therapeutic efficacy, because MSCs are considered to reach tumors including metastatic lesions and to deliver therapeutic molecules in a concentrated fashion. This targeted therapy can also reduce systemic adverse side effects, because the anti-cancer agents act locally at the site of tumors without elevating their systemic concentrations. In the present study, we developed genetically-modified MSCs that produce retroviral vectors encoding HSVtk, aiming at augmenting therapeutic efficacy of systemic suicide cancer gene therapy. The tumor tropism and anti-tumor effects of vector-producing MSCs (VP-MSCs) were examined by intravascular injection in tumor-bearing nude mice. MSCs isolated from the bone marrow of SD rats were transfected with plasmid DNA expressing luciferase alone (=non-VP-MSCs) or whole retroviral vector components (LTR-Luc or LTR-HSVtk with Gag-pol and VSV-G) (=VP-MSCs) by nucleofection. To assess tumor tropism of MSCs, nude mice were subcutaneously inoculated with 9L rat glioma cells or Rat-1 fibroblasts, and were subsequently injected with luciferase-expressing MSCs through the left ventricular cavity. The transgene expression was periodically traced by using an in vivo imaging system. As a result, the transgene expression accumulated at the site of subcutaneous 9L tumors, but undetectable at the site of Rat-1 fibroblasts. In addition, the injection of luciferase-expressing VP-MSCs caused much stronger signal of bioluminescence at the site of 9L tumors compared with luciferease-expressing non-VP-MSCs. Immunostaining study showed that luciferase-positive cells (injected MSCs and transduced glioma cells) were detected at the periphery of tumors. To evaluate the therapeutic efficacy, tumor-bearing nude mice were treated with non-VP-MSCs or VP-MSCs combined with HSVtk/GCV system and then the size of subcutaneous tumors was periodically measured. In this model experiments, tumor growth was more efficiently suppressed by injecting VP-MSCs compared with non-VP-MSCs. The present study suggests the effectiveness of VP-MSCs in suicide cancer gene therapy. The therapeutic benefit of this strategy should be further examined in orthotopic and metastatic tumor models.
Mesenchymal Stromal Cells (MSCs) Acquire Cardiac-Specific Markers but Do Not Become Functional Cardiomyocytes and Retain the Stromal Phenotype.
Authors: Rose Robert A. , Jiang Huije , Wang Xing Hua , Helke Simone , Tsoporis James N. , Gong Nanling , Keating Stephanie C.J. , Parker Thomas G. , Bacx Peter , Keating Armand
Published: April 2020

Although marrow-derived MSCs may be beneficial in treating models of ischemic heart disease, their ability to transdifferentiate into functional cardiomyocytes remains unclear. MSCs from female transgenic mice expressing green fluorescent protein (GFP) under the control of the cardiac-specific α-myosin heavy chain promoter were co-cultured with male rat embryonic cardiomyocytes (rCMs). This novel in vitro model permits identification and functional analysis of putatively transdifferentiated MSCs based on GFP expression. In co-culture, 6.3% of MSCs became GFP+. Quantitative PCR using murine-specific primers revealed that co-cultured MSCs express the cardiac-specific genes atrial natriuretic factor, Nkx2.5 and α-cardiac actin. Furthermore, immunohistochemistry on GFP+ MSCs established co-expression of the sarcomeric proteins troponin I and α-actinin, but without a clear sarcomeric pattern. Despite expression of cardiac genes, patch-clamp experiments illustrate that GFP+ MSCs did not fire action potentials and did not express voltage gated sodium and calcium currents (INa and ICa). In contrast, action potentials, INa and ICa were readily observed in all GFP− rCMs studied in the co-cultures. When investigated using slow voltage ramps, GFP+ MSCs displayed electrical properties typical of non-excitable cells, suggesting retention of a stromal cell phenotype. Indeed, detailed immunophenotyping of GFP+ MSCs demonstrated expression of all antigens used to characterize MSCs as well as the acquisition of additional markers of cardiomyocytes with the phenotype: CD45− CD34+ CD73+ CD105+ CD90+ CD44+ SDF1+ CD134L+ collagen type IV+ vimentin+ troponin T+ troponin I+ a-actinin+ connexin 43+. Although cell fusion between rCMs and MSCs was detectable by fluorescence in situ hybridization, the very low frequency (0.68%) cannot account for the phenotype of the GFP+ MSCs. In conclusion, we have identified a MSC population that shows plasticity towards the cardiomyocyte lineage but that retains mesenchymal stromal cell properties, including a non-excitable electrophysiological phenotype. The demonstration of a MSC-derived population co-expressing cardiac and stromal cell markers may explain the conflicting results in the literature regarding transdifferentiation and indicates the importance of extensive immunophenotypic and functional analysis of the manipulated cells. The data also infer the need to identify mechanisms other than cardiomyocyte differentiation that underlie the effects of MSCs on myocardial injury.
Isolation of Mesenchymal Stem Cells from Human Bone Marrow Using Aldehyde Dehydrogenase as a Marker.
Authors: Nishimoto Eishi , Yamada Yasuhiro , Miyake Hirosada , Mitsuya Hiroaki , Yonemura Yuji
Published: April 2020

Mesenchymal stem cells (MSCs) represent multipotent stem cells that are capable of self-renewal while maintaining their multipotence and can differentiate into a variety of cell types, including osteoblasts, chondrocytes, myocytes, adipocytes, and beta-pancreatic islets cells. However, the origin and properties of human MSCs remain to be elucidated. Aldehyde dehydrogenase (ALDH) is known to be useful for isolation of hematopoietic stem cells as well as neural stem cells and endothelial progenitors. In this study, we attempted to isolate MSCs from human bone marrow (BM) using ALDH as a marker for MSCs. Mononuclear cells (MNCs) in BM were first sorted to ALDHnegative, ALDHlow, and ALDHhigh cell fractions. ALDHnegative and ALDHlow cells had no colony forming units-fibroblast (CFU-F). ALDHhigh cells represented 1.8±0.8% of MNCs in human BM (n=4) and contained CD45 negative cells (9.9±5.3%). When ALDHhigh CD45+ cells and ALDHhighCD45− cells were cultured with MF start® (TOYOBO, Osaka, Japan), CFU-F was found in the latter cells but not in the former cells. The frequency of CFU-F in ALDHhighCD45− cells and MNCs in BM was 1 in 330 and 13,600, respectively. We found that CFU-F differentiated to adipocytes and osteocytes as examined using the human mesenchymal stem cell functional kit (R&D, Minneapolis) and immunological staining with FABP-4- and osteocalsin-specific antibodies. We also tried to isolate MSCs in human cord blood (CB) and granulocyte-colony stimulating factor (G-CSF)-mobilized peripheral blood (PB). Proportion of ALDHhigh cells was 2.1±0.8% in CB (n=3) and 0.6±0.2% in G-CSF-mobilized PB (n=4). CB ALDHhigh cells contained CD45− cells (14.9±2.8%), but G-CSF-mobilized PB ALDHhigh cells contained only few CD45− cells (1.3±0.8%). CFU-F was seen in CB, while no CFU-F was identified in G-CSF-mobilized PB. The current data demonstrate that ALDHhighCD45− cells can differentiate into adipocytes and osteocytes and that ALDH is a useful marker for isolation of human MSCs.
Treatment of Philadelphia Positive Chronic Myeloid Leukemia with Imatinib: Introducing the Concept of Confirmed Molecular Response.
Authors: Palandri Francesca , Rosti Gianantonio , Iacobucci Ilaria , Testoni Nicoletta , Castagnetti Fausto , Breccia Massimo , Abruzzese Elisabetta , Martino Bruno , Cilloni Daniela , Pane Fabrizio , Gobbi Marco , Intermesoli Tamara , Stagno Fabio , Capucci Adele , Martinelli Giovanni , Baccarani Michele
Published: April 2020

Background: Imatinib (IM) is the drug of choice for the treatment of CML, where it was shown that a complete cytogenetic response (CCgR) and a major molecular response (MMolR) at 12 months (mos) were the best surrogate markers of progression-free survival (PFS) (O’Brien et al, NEJM, 2003; Hughes et al, NEJM 2003). The issue of the MolR is particularly sensitive, because the degree of the MolR may fluctuate over time and among different labs, also for methodological reasons (Hughes et al, Blood 2006).

Aim: To evaluate the prognostic value of the MolR based on a retrospective analysis of the patients (pts) who achieved a CCgR with IM 400 mg daily.

Patients and Methods: 130 pts who achieved a CCgR for more than 1 year (confirmed CCgR) and were tested for MolR at least 3 times after achieving the CCgR, were analyzed. Cytogenetic response was assessed every 6 mos by conventional analysis of at least 20 marrow cell metaphases. MolR was assessed every 3 mos by RTQ-PCR (TaqMan) on peripheral blood buffy coat cells and was defined as MMolR when BCR-ABL:ABL<0.05%, corresponding to 0.1% on the International Scale.

Results: In 71/130 pts (55%) MolR was always major (stable MMolR). In 19/130 (15%) pts MolR was sometimes major and sometimes less than major (unstable MMolR). In 40/130 pts (30%) MolR was never major (never MMolR). The proportion of pts remaining in CCgR after 5 years was calculated by the Kaplan-Meyer method (Fig. 1) and was 95% for pts in stable MMolR, 77% for pts with unstable MMolR and 62% for pts without a MMolR during follow-up (p<0.0001, log-rank test).

Conclusions: These data confirm that achieving a MMolR is prognostically important but point out that the prognostic value of achieving a MMolR is greater if the response is confirmed, and introduce the concept of sustained Mol response, alerting from relying on single determinations.

Figure Figure
Relevance of HLA-C Epitopes C1 and C2 for the Survival of Patients with AML and CML after Allogeneic Blood Stem Cell Transplantation.
Authors: Bruns Ingmar , Czibere Akos G. , Uhrberg Markus , Enczmann Jürgen , Kobbe Guido , Neumann Frank , Wernet Peter , Haas Rainer , Fischer Johannes C.
Published: April 2020

BACKGROUND HLA-C epitopes can be grouped in C1C1, C1C2 or C2C2 ligands and mediate NK cell dependent immune response. Especially in haploidentical allogeneic stem cell transplantation a HLA-C ligand mismatch improves event free survival (EFS) in patients with AML known as Velardi effect. Recently, we could show in 109 CML patients that those with a C1C1 phenotype showed better overall survival (OS) and lower rates of treatment related mortality (TRM) (Fischer JC et al. J Immunology. 2007). But, the role of HLA-C ligands in allogeneic transplantations remains controversial.

PATIENTS AND METHODS In this study we retrospectively analyzed a group of 88 patients with AML or CML (n=34), MDS (n=21) or lymphoid malignancies (Non-Hodgkin-Lymphoma or ALL) (n=31) receiving unrelated allogeneic blood stem cell transplantation after myeloablative and non-myeloablative conditioning regimens. HLA-C alleles were determined by DNA-based direct sequencing of all donors and recipients included into this study.

RESULTS Looking at the group of 34 patients with AML or CML, the 13 recipients with a C1C1 phenotype showed increased OS compared to those with C1C2 and C2C2 phenotypes (all patients alive with a median follow-up of 154 days, range 90 to 665 days vs. a mean survival of 381 days, respectively; p=0.049). All recipients with a C1C1 phenotype received grafts with matched HLA-C alleles. Within the subgroup of patients with C1C2 or C2C2 phenotypes 6 patients had a HLA-C mismatch which was associated with significantly (p=0.016) increased OS (all patients alive with a median follow-up of 575 days, range 133 to 899) compared to matched HLA-C phenotypes (median survival of 254 days). In recipients with C1C1 phenotype the risk for TRM following HLA-C matched hematopoietic stem cell transplantation was reduced as reflected by an odds ratio of 0.13. In turn, the group receiving HLA-C mismatched grafts had a lower incidence of relapse. This effect was independent from the direction of the mismatch, graft vs. host or host vs. graft. The effects described above were not observed in patients with MDS, ALL or lymphoid malignancy.

CONCLUSION The beneficial effects of a C1C1 HLA-C phenotype could be confirmed for patients with CML and AML in our patient cohort. Our data also suggest that patients with myeloid malignancies and an unfavourable C1C2 or C2C2 HLA-C phenotype benefit from a donor with HLA-C ligand mismatch.
Drug Metabolism Gene Polymorphisms and the Risk of Hepatic Sinusoidal Obstruction Syndrome in Children with Inherited Diseases Receiving Haematopoietic Stem Cell Transplantation.
Authors: Fernandes Juliana F. , Rocha Vanderson , Porcher Raphael , Neven Benedicte , Hue Christophe , Tavela Marli , Fischer Alain , Zago Marco Antonio , Cavazzana-Calvo Marina
Published: April 2020

Hepatic Sinusoidal Obstruction Syndrome (SOS) following high dose cytotoxic therapy remains a serious complication after heamatopoietic stem cell transplantation (HSCT). Genetic polymorphisms that interfere with metabolism of drugs used in conditioning regimens have been associated with SOS after HSCT. In order to study this association in children given a HSCT for inherited diseases of the heamatopoietic system we have analyzed candidate genes in 99 children: 35 who developed SOS and in 64 children who did not develop SOS; both groups were transplanted in the same center and matched for year of transplant (from 1998 to 2004). Four families of candidate genes were analyzed: P450 cytochrome [CYP2B6*2(C64T), *3(C777A), *4(A785G), *5(C1459T), *6(G516T)], Glutathione-S-Transferases [GSTM1 (null), GSTT1 (null), GSTP1 (A313G)], Methylenetetrahydrofolate reductase [MTHFR (C677T)] and Vitamin D receptor [VDR (ApaI, TaqI and BsmI)]. The most frequent diagnosis was severe combined immunodeficiency. The two groups had no statistical differences for gender, diagnosis, type of transplant and source of stem cells. Conditioning regimen consisted in busulfan and cyclophosphamide in 91% of SOS patients and 87% of non-SOS patients (p=ns). There was no statistical difference on the frequency of all gene polymorphisms studied in both groups of patients, except for the gene CYP2B6*2 (C64T). In fact, the mutant allele T (genotypes CT or TT) was present in 31% of SOS patients and 9% of remainders (p=0.01). CYP2B6 is known to play an important role in the metabolism of chemotherapeutic agents such as cyclophosphamide (CY). In conclusion, we found that the polymorphism in CYP2B6*2 allele of the P450 cytochrome family is more frequent in children with inherited diseases who have developed SOS after HSCT than in those who did not develop SOS. This finding may define better surveillance and prophylaxis of SOS for children with the mutant allele of CYP2B6*2 given a HSCT.
Genetic Polymorphisms of Gluthatione-S-Transferases in Two Different Populations of Fanconi Anaemia Patients.
Authors: Fernandes Juliana F. , Rocha Vanderson , Porcher Raphael , Bittencourt Marco , Santos Moema N. , Skvortsova Yulia , Bonfim Carmen , Tavela Marli , Socie Gerard , Gluckman Eliane , Pasquini Ricardo , Zago Marco Antonio
Published: April 2020

Fanconi anaemia (FA) patients are known to have a markedly increased toxicity of chemotherapeutic agents, due to impairment in various important cellular processes, such as DNA repair. This toxicity had led to several alterations in preparative regimens in the setting of haematopoietic stem cell transplantation (HSCT). Several polymorphisms in genes coding for metabolising enzymes and drug transporters have been described and are now being associated with predisposition to efficacy or toxicity to different drugs. We have driven the hypothesis that FA patients may have a different frequency of those gene polymorphisms. For this purpose we have studied two different FA populations and their controls. The following of genes were analyzed: P450 cytochrome [CYP2B6*2(C64T), *3(C777A), *4(A785G), *5(C1459T), *6(G516T)], Glutathione-S-Transferases [GSTM1 (null), GSTT1 (null), GSTP1 (A313G)], Methylenetetrahydrofolate reductase [MTHFR (C677T)] and Vitamin D receptor [VDR (ApaI, TaqI and BsmI)]. The first groupt consisted of 57 FA patients, from Saint Louis Hospital in Paris compared with 107 samples of a normal control population (healthy marrow donors) of the same ethnic group. The second group of patients consisted of 72 FA patients treated in the Hospital de Clinicas in Curitiba (Brazil) compared to a control population of 72 healthy blood donors matched to age, sex and ethnicity.

Results: in both groups, the frequency of CYP2B6 alleles, MTHFR, VDR and GSTP1 polymorphisms was not statistically different between patients and controls. For GSTT1, gene deletions were found in 5 (9%) of 57 FA patients and in 27 (25%) of the 107 controls from the first group, which revealed a significant difference (p=0.013). This result was also found in the second group of patients. In fact, GSTT1 null genotype was present in 8 (11%) of the 72 FA patients and in 19 (26%) of the normal controls (p=0.016). For GSTM1, the first group had no statistical difference in the frequency of gene deletions between FA patients and their controls. However, in the second group, GSTM1 null phenotype was found in 38 (53%) patients and in 26 (36%) controls (p=0.05). In conclusion, we found in two ethnically different groups, that FA patients may have a lower frequency of GSTT1 null genotype, compared to a normal population. GSTM1 null phenotype had a higher incidence in FA patients in the Brazilian group, but was not reproducible in the French group. This finding should be further analyzed to explain if it can be related to the disease itself and/or if it translates in increased toxicity of drugs in FA patients.