bluuebunny/crossref_metadata_2025 huggingface dataset:
Immunotherapy with Rituximab after Peripheral Blood Stem Cell Transplantation for Multiple Myeloma.
Authors: Massumoto Celso Mitsushi , Junior Edilson Pinheiro , Baiocchi Otávio C.G. , Pinheiro Ronald , Alves Adelson
Published: April 2020

Introduction: autologous stem cell transplantation is a potentially curative or may augment the time to progression in Multiple Myeloma (MM) patients. The immunotherapy with rituximab may help control the minimal residual disease after high dose chemotherapy. Twenty percent (20%) of Multiple Myeloma patients express the CD20+ protein and can be target for immunotherapy.

Objective: The aim of this study was to evaluate the use of rituximab after autologous stem cell transplantation for Multiple Myeloma.

Patients and Methods: eight patients (4 male) with a median age of 53 (range 43–59) years diagnosed with MM. All of them had received at least one previous regimen were enrolled in the protocol study. All patients signed the consent form. Patients in relapse received a salvage regimen with C-VAD n=2 (cyclofosfamide 4 g/m2 e vincristine 0.4 mg/d (d 1–4), doxorrubicin 0.9 mg/m2 (d1-4) e dexametasone 40 mg (d1-4; 9-12; 17–22) or cyclofosfamide (1OO mg/kg, n=7) followed by stem cell harvesting. The preparative regimen was Busulfan 12 mg/kg and cyclofosfamide 120 mg/kg or Melphalan 200mg/m2. Rituximab at a dose of 375mg/m2 weekly x 4 was given every 6 months for 2 years after SCT. The clinical characteristics of the patients are shown on Table 1.

Results: the median time to ANC and platelets engraftment was 11 (range 8–12) and 26 (range 17–35) days. Patients have been in CR at a median time of 11 months follow-up. Minor Rituximab-associated toxicities were seen:rigor, fever and short of breath that were controlled with acetaminophen and diphenidramine.

Conclusion: the Rituximab given after autologous stem cell transplantation is safe in Multiple Myeloma patients and may prolong time to disease progression. A randomized study is required to evaluate the role of rituximab after ASCT.

Table 1 - Clinical Characteristics of Patients

Patients Age/gender Status Pre- BMT Status Post- BMT Salvage Tx Prep. regimen ANC/Platelets X1000 MM3/ml Follow-up (months) FRC 57/M PR PR C-VAD BU+MEL 12/28 EXPIRED MB 52/F CR1 CR1 C-VAD BU+MEL 12/60 EXPIRED AM 52/F PR PR C-VAD BU+MEL 9/26 EXPIRED IM 54/M PR CR Cyx2 BU+MEL 12/21 ALIVE GAD 50/F PR CR Cyx2 BU+MEL 12/35 ALIVE SCM 59/M CR CR Cyx2 BU+MEL 10/17 ALIVE MAD 63/F CR CR C-VAD MELPHALAN 12/25 ALIVE JFC 51/M CR CR C-VAD BU+MEL 12/18 ALIVE
Autologous Bone Marrow Transplantation for Follicular Lymphoma in First Remission: Long Term Follow up of Two Sequential Trials with Standard Dose and High Dose CHOP Induction.
Authors: Freedman Arnold S. , Gribben John G. , Neuberg Donna , Li Sigui , Kim Helen , Mauch Peter , Nadler Lee M.
Published: April 2020

High dose therapy and autologous stem cell transplantation (ASCT) has been employed as salvage therapy for patients (pts) with relapsed follicular non-Hodgkin’s lymphoma (FL). Recently, the randomized European CUP trial demonstrated that following 3 cycles of CHOP, ASCT significantly improved both disease free and overall survival when compared to continued CHOP in pts with relapsed FL. In an attempt to improve the results of high dose therapy in FL, we undertook 2 sequential studies for pts with FL in first remission. In the first study, 77 pts with previously untreated advanced stage FL (median age 43), received 6–8 cycles of standard dose CHOP (SD-CHOP) + involved field radiotherapy followed by high dose chemoradiotherapy and anti-B cell purged autologous bone marrow transplantation (ABMT). Following SD-CHOP induction, 27 of 77 pts (36%) were in CR and 50 in a minimal disease state (< 2 cm masses, < 20% BM involvement). At BM harvest, 36 pts had histologic evidence of BM involvement. In the second trial, 19 pts with advanced stage disease (median age 44) were treated with 4 cycles of dose intensified CHOP (HD-CHOP) (cyclophosphamide 1.5 g/m2 day 1 and 2) with G-CSF support followed by high dose chemoradiotherapy and anti-B cell purged autologous ABMT. Following HD-CHOP, 13 of 19 pts were in CR. At BM harvest, 7 of the 19 HD-CHOP pts had histologic BM involvement. Following ABMT, there were 2 acute in-hospital deaths in the pts receiving SD-CHOP induction and none in the pts who received HD-CHOP induction. Nine late deaths in remission were observed in the SD-CHOP pts including 6 from MDS/AML, and 2 late deaths in the HD-CHOP pts. Thirty-three pts who received SD-CHOP remain alive without relapse, with a median follow up of 12 years. Ten pts who received HD-CHOP induction remain alive without relapse, with a median follow up of 9.1 years. For pts receiving SD-CHOP induction, the DFS and overall survival at 10 years are 42% (90% CI: 33%–52%) and 64% (90% CI: 55%–73%), respectively. For pts who received HD-CHOP induction, the DFS and overall survival at 10 years are 59% (90% CI: 38%–79%) and 75% (90% CI: 57%–93%), respectively. The impact of BM treatment and DFS was examined. Pts with known bcl-2 translocations for whom post-BM purging samples were available for pcr examination were analyzed. Following ABMT, pts who were reinfused with pcr negative BM had a significantly better DFS than the pts who were reinfused with pcr positive BM. This study suggests that a subset of pts with FL experience long term remission following ABMT in first remission.
Selective and Efficient Gene Delivery of CD40-Ligand by a Fiber-Modified Adenovirus Vector with Specific Antibody to Human Leukemia and Myeloma.
Authors: Kato Kazunori , Hirai Sachie , Masuta Yukari , Kuroishi Aiko , Nakamura Kiminori , Hamada Hirofumi
Published: April 2020

The use of adenovirus vector for cancer gene therapy is limited by their low transduction efficiency for lymphoma, leukemia and myeloma. We previously reported that highly efficient gene delivery of CD40-ligand by a modified adenovirus vector with the integrin-binding motif, RGD, in the H1 loop of the fiber knob (AxCAhCD40L-F/RGD) could induce phenotypic alteration followed by T cell immune response to autologous leukemia cells. But the utility of adenovirus with RGD-motif is still limited by their lack of specificity on tumor cells. Recent studies revealed a novel strategy of targeting adenovirus using a bispecific single-chain antibody (scFv) specific for adenovirus and target molecules on tumor cell surface. However, this approach should permit the production of high quantities of active bispecific scFv for in vivo use. To target adenovirus to hematopoietic tumor cells efficiently, we herein constructed a modified adenovirus vector that contained a synthetic immunoglobulin G-binding domain (termed Z33) in H1 loop of the fiber knob. A recombinant adenovirus encoding EGFP, lacZ (as reporter gene; Ax3CAZ3-F/Z33 or Ax3EGFP-F/Z33) or CD40L (as a therapeutic gene; Ax3CD40L-F/Z33) with Z33-modified fiber were tested for gene transfer efficiency into human tumors such as lymphoma, leukemia and myeloma cells. By the treatment with various antibodies specific for CD20 (Rituximab), CD40, CD38, CCR2 or CXCR4 that are expressed on leukemic cells, we achieved 3 to 10-fold enhancement of gene expression in lymphoma/leukemia (Ramos, Daudi or THP-1) and myeloma cells (MM1S, IM-9 or KMS5) compared with control IgG-treated tumors. We also examined specific gene delivery to freshly isolated leukemia B cells from patients that also contains normal lymphocytes. By using antibody to CD20 or CD40, we could selectively deliver CD40L gene with Ax3CD40L-F/Z33 into leukemia B cells (>50% at 300 pu/cell), but not in T and monocytes, followed by the induction of immune costimulatory molecules that are important in anti-leukemia immune response. Overall, our results indicated that combination of Z33-modified adenovirus vector and tumor specific antibody can be used as a modality for the gene therapy of leukemia and myeloma.
Development of Novel Multigene Lentiviral Vectors.
Authors: Chinnasamy Nachimuthu , Milsom Michael , Neuenfeldt James , Shaffer James , Margison Geoffrey P. , Fairbairn Leslie J. , Chinnasamy Dhanalakshmi
Published: April 2020

Lentiviral vectors are efficient in transducing dividing and non-dividing cells. Many gene transfer applications require vectors that express more than one protein which include but not limited to therapeutic gene plus a selectable marker gene, multiple genes encoding different subunits of a complex protein or multiple independent genes that cooperate functionally. Previous strategies in constructing multigene lentiviral vectors by other groups included splicing, multiple internal promoters, internal ribosome entry site (IRES), and fusion proteins. In this study we explored the feasibility of expressing homeobox B4 (HOXB4), O6-methylguanine-DNA-methyltransferase (MGMT), and enhanced green fluorescent protein (EGFP) under the control of a single promoter with the use of IRES sequence from encephalomyocarditis (EMCV) virus and/or foot and mouth disease virus (FMDV) 2A cleavage factor in a HIV-1 based self-inactivating lentiviral vector. In bicistronic vectors the small FMDV 2A sequence can be used efficiently as an alternative to IRES. In case of tricistronic vectors the 2A sequence can be used in combination with IRES to obtain simultaneous expression of three cDNAs under the control of a single internal promoter. Monocistronic, bicistronic and tricistronic vectors were constructed and a three-plasmid expression system was used to generate vesicular stomatitis virus glycoprotein pseudotyped lentiviral vector particles. Viral titers were measured by quantification of HIV p24 gag by ELISA. The relative efficiency of transgene expression in transduced cells by various vectors was compared by appropriate methods including fluorescence microscopy, flow cytometry, immunocytochemistry, biochemical assay, and western blotting. All the multigene vectors produced high titer viral particles and were able to simultaneously express two or three transgenes in transduced cells. However, the expression of EGFP from monocistronic vector as measured by mean fluorescence intensity was 3–8 times and 10–20 times higher than that of bicistronic and tricistronic vectors respectively. Notably expression of second gene encoded by the bicistronic vector containing FMDV 2A was 2–3 fold higher than that of IRES-based vector. Expression of MGMT in monocistronic and bicistronic constructs was also 4 to 20 times higher than tricistronic vectors. The FMDV 2A cleavage factor efficiently mediated the generation of the expected cleavage products from the artificial fusion protein. These vectors can be used in a wide range of applications including the expression of: multiple subunits of a functional protein, tumor antigen and co-stimulatory molecule(s), multiple tumor antigens for immunotherapy applications, and co-expression of selectable marker gene/s with therapeutic gene/s.
Activation-Dependent Expression of an Interleukin-2 Transgene Mediated by Transduction with a Lentiviral Vector Enhances T Cell Functions.
Authors: Sun Qi , Chorney Karen , Stine Carol , Lucas Kenneth G.
Published: April 2020

Adoptive T cell immunotherapy (ATCI) is an evolving strategy that explores antigen-specific T cells manipulated ex vivo as therapeutic agents. Although the concept of ATCI has been tested clinically, with success in the treatment of post-transplant EBV induced lymphoproliferative disease, one of the major obstacles hindering its application to other malignancies is the procurement of tumor-specific T cells that possess potent anti-tumor functions even in the inhibitory environment at the tumor sites. This study aims to genetically engineer enriched viral specific T cells for improved immune functions. A self-inactivating lentiviral vector (SIN) CD69p-IL2 was constructed to encode the transgene interleukin-2 (IL2) under the control of a human CD69 promoter (CD69p), and this vector was tested in ex vivo cultivated EBV-specific T cells. SIN vector allows a high degree of autonomy for the internal promoter, and CD69 expression in the T cells is closely associated with T cell activation. Experiments showed that the SIN vectors efficiently transduced EBV-specific T cells, both CD4 and CD8. Furthermore, the newly cloned CD69p exhibited a higher degree of responsiveness to physiological antigen stimulation than the early promoter from the cytomegalovirus (CMVp). In response to stimulation by EBV-infected B cells, the percentage of IL2 expressing cells was 2 fold higher for the activated CD69p-IL2 transduced T cells than the non-transduced, or the CMVp-IL2 transduced, counterparts. In correlation with the stronger IL2 expression, 3 fold more T cells expressed the anti-viral cytokine interferon-γ (IFN-γ) in the CD69p-IL2 transduced T cells than the CMVp-IL2 transduced, and the IFN-γ expression at the single cell level was 2 fold higher in the former, indicating an enhanced functionality. While the culture supernatant from the CMVp-IL2 transduced T cells contained IL2 at a concentration 2000 fold higher than the non-transduced T cells, the IL2 level in the media from the CD69p-IL2 transduced T cells was comparable to that in the control, suggesting the IL2 expression mediated by the CD69p more relevant to T cell functions than the CMVp. These results may serve as a foundation for the further development and clinical application of specific T cells engineered for enhanced immune functions.
Clinical Significance of Symptom Burden and Quality of Life Decrement in Cancer Patients with Mild Anemia.
Authors: Schwartzberg Lee S. , Fortner Barry V. , Houts Arthur C.
Published: April 2020

Background. Cancer related anemia (CRA) is associated with increased symptom burden and reduced quality of life (QoL) compared to non-anemic subjects. Standard treatment guidelines for CRA recomend treatment when hemoglobin (Hb) declines to ≤ 10 g/dl. We compared patients with mild anemia to those with normal Hb level to assess clinical significance of mild anemia. Methods. 3416 patients from a large community oncology database sorted by gender and Hb were retrospectively reviewed.

Patients receiving chemotherapy (<30 days) and/or growth factor (< 60 days) were excluded. Each case provided one Hb determination and same day self reported scores on the Cancer Care Monitor (CCM), a validated measure of symptom burden, functioning, and health related QoL comprising 6 scales. All CCM items are rated for symptom severity on a 0–10 scale. Effect sizes for male groups are calculated relative to normal males (Hb > 14), and effect sizes for females are calculated relative to normal females (Hb > 12). Positive effect size (Cohen’s exact d) values indicate greater symptom burden, and negative effect size values indicate lower quality of life.

Results. Group differences on CCM measures were not accounted for by demographic, cancer diagnosis, and chemotherapy history differences. Table 1 shows a pattern of greater symptom burden, lower functioning, and worse QOL for males and females with mild anemia (p < .05). As compared to standards for minimal clinically important differences such as found with SF-36 (0.09 < Cohen’s d < 0.28). patients with mild anemia showed clinically significant differences in terms of effect size (0.11 < Cohen’s d < 0.61).

Conclusions. The QoL impact of mild CRA is significant and failure to treat mild anemia may result in unnecessary symptom burden and noteworthy decrements in health related quality of life.

Differences in QoL for Patients with Mild Anemia

M Hb12<14 M Hb >14 Effect Size F Hb 10<12 F Hb >12 Effect Size Cancer Care Monitor Measure* (n = 327) (n = 471) Males (n = 449) (n = 2169) Females Note. *Mean (SE) for each item or scale. Adjusted means with different subscripts across rows are significantly different (p < .05) by Bryant Paulson comparisons. Fatigue Item 3.10 (0.16) b 2.27 (0.13) c 0.30 3.47 (0.13) b 2.65 (0.06) d 0.30 Physical Symptoms 49.54 (0.58) b 45.49 (0.48) c 0.38 49.32 (0.49) b 46.97 (0.22) d 0.22 General Distress 49.51 (0.57) b 47.07 (0.47) d 0.19 51.23 (0.49) c 49.85 (0.22) b 0.11 Despair 50.74 (0.52) b 48.58 (0.43) c 0.25 50.37 (0.44) b 49.13 (0.19) c 0.14 Impaired Performance 55.55 (0.87) b 49.78 (0.70) c 0.61 55.02 (0.73) b 49.90 (0.32) c 0.48 Quality of Life 49.41 (0.79) b 53.46 (0.64) c −0.45 48.95 (0.67) b 52.39 (0.29) c −0.35
Resource Utilization, Costs and Type of Malignancy Associated with Prolonged Length of Stay in Autologous Stem Cell Recipients.
Authors: Patchett Linda J. , Hill John M. , Fitzmaurice Thomas F. , Meehan Kenneth R.
Published: April 2020

In order to contain costs, MDs must first identify the clinical factors contributing to increased resource utilization associated with an autologous stem cell transplant.

We performed a retrospective clinical and cost analysis of all autologous transplants performed at Dartmouth- Hitchcock Medical Center over a 30 month period (2002-2004) and identified patients who had a prolonged length of stay > 25 d (PLOS). We pinpointed the clinical characteristics and hospital course of each patient to identify trends. The hospital cost-accounting system highlighted resource utilization and costs of the transplants, allowing a comparison between patients with a PLOS and all other transplant patients.

PROLONGED LENGTH OF STAY (PLOS)

Results: All Patients LOS < 25 days LOS > 25 days # of patients 87(100%) 58 (67%) 29 (33%) LOS (days) Mean (Median) 24 (22) 20 (20) 31 (31) DISEASE (n = no. of patients) AML 14 9 5 HD/NHL 44 24 20 MM 28 25 3 Other (ITP) 1 1 ENGRAFTMENT (median) ANC > 500 (Platelets > 20K) 12 (18) 11 (16) 13 (27) TRANSFUSIONS UNITS /PT (median) RBC /Platelets > 20 4 (3) 3 (2) 7 (7) PARENTAL NUTRITION (TPN) # of days (median) 9 6 14 TOXICITIES >= GRADE 3 NCI (Common Toxicitity Criteria) Nausea and Vomiting 36% 77% Diarrhea 9% 45% Mucositis 36% 41% Anorexia 57% 83% INFECTION RATE 10% 34% ICU TRANSFER 3% 3%

Major contributors to costs included nursing/daily room charge costs (39%), pharmacy (39%), Blood Bank (6%), Laboratory (12%), and other costs (3%). The average daily costs are $4252.

The PLOS cohort had grade > 3 toxicity, increased infection rate, engrafted later and required more transfusional support. 1 pt was transferred to the ICU for temporary management. Of the 29 patients identified with PLOS, none died and all were discharged from the hospital. 45% of NHL/HD patients experienced a prolonged LOS, representing 68% of the PLOS cohort. The median LOS<25d is 20d and the median LOS>25d is 30.5d. At an average daily cost of $4252, these additional 10.5 days of hospitalization costs are substantial. Based on these findings, identification of factors underlying PLOS in the NHL/HD cohort may provide the key to minimizing cost of autologous stem cell transplant. Accordingly, we are assessing the impact of age, number of pre-transplant treatment regimens, number of peripheral blood stem cells reinfused, use of IL-2 for post-transplant immune modulation, and the day 15 absolute lymphocyte count on LOS in this population.
Gelclair® for the Treatment of Chemotherapy-Induced Stomatitis in Transplant and Hematology Patients: An Interim Analysis.
Authors: Liewer Susanne E. , Hecht Keith A. , Smith Courtney F. , DeRemer David L. , Howard Dianna , Brown Randy A. , Herzig Roger H. , Adams Val R.
Published: April 2020

Patients receiving treatment for hematologic malignancies (HM) commonly experience stomatitis. While there is no standard therapy, pain management and infection prophylaxis are the cornerstones of treatment. Gelclair® a bioadherent gel has been shown to decrease pain associated with stomatitis but has yet to be evaluated in this patient population. This open label, single-arm study evaluates the effectiveness of Gelclair® administered every 8 hours to relieve pain associated with treatment-induced stomatitis in patients with HM. Efficacy was determined by obtaining hourly pain scores on a 10-point scale for the first 8 hours, then every 6 hours until resolution of symptoms, while controlling for stomatitis severity and opioid consumption. Secondary endpoints included quality of life [functional living index-cancer (FLIC)], ability to consume a soft diet, and ability to take oral medications. Fifteen of 30 planned patients have been enrolled, with 12 being transplant patients (8 auto, 4 allo). Melphalan was the most common therapy received and multiple myeloma was the most common patient diagnosis. The median time on study was 5 days. The median stomatitis severity was grade 2 with a range of 1 to 3 (NCI criteria). The mean pain score at baseline was 5.8 +/− 0.6, which declined to 3.8 +/− 0.6 one hour after Gelclair® application and remained below baseline for 8 hours. Over the first 72 hours, average daily pain scores, stomatitis severity, and opioid consumption all increased, while WBC decreased. The average FLIC score at baseline was essentially unchanged on day 1, 7 or at the end of study (102, 102, 98, and 101, respectively). Oral meds were administered and soft diet tolerated during 93% and 85% of treatment days. Adverse events were limited to nausea with or without vomiting in 2 patients. Interim analysis suggests Gelclair® improves pain scores quickly while allowing patients to continue oral medications. This improvement is sustained throughout the dosing interval. Data from all evaluable patients will be presented at the meeting.
Homodimerization of Retinoic Acid Receptor alpha through its Fusion Partners Underlies Pathogenesis of Acute Promyelocytic Leukemia.
Authors: Kogan Scott C. , Phan Vernon T. , Sternsdorf Thomas , Maunakea Mei Lin , Sohal Jastinder , Silletto Angela , Galimi Francesco , Evans Ronald M.
Published: April 2020

In almost all cases of acute promyelocytic leukemia (APL), t(15;17)(q22;q12) fuses the promyelocytic leukemia (PML) gene with the retinoic acid receptor alpha (RARA) gene. In rare cases of APL, other genes are fused to RARA including PLZF, NPM, NuMA, and STAT5b. These chimeras are collectively referred to as X-RARA fusions. Common features of the X-RARα fusions include their ability to form dimers through the X domain proteins while retaining the ability to interact with rexinoid receptors (RXRs, the proteins with which RARα normally heterodimerizes). We previously created an artificial X-RARα: p50-RARα (a fusion of the dimerization interface of p50 NFκB with the portion of RARα found in naturally occurring X-RARα proteins). p50-RARα has effects similar to PML-RARα including (i) enhancing repression at retinoic acid response elements, due to impaired release of co-repressors at low levels of all-trans retinoic acid (ATRA), and (ii) inhibiting differentiation of myeloid cell lines. We generated transgenic mice expressing p50-RARα under the control of the MRP8 promoter. p50-RARα had a minimal effect on myelopoiesis and initiated myeloid leukemias at a rate of <5% in the first year of observation. Nevertheless, transduction of p50-RARA transgenic bone marrow with a retrovirus expressing an activated cytokine receptor (βcV449E) generated leukemias with features of APL including therapeutic responsiveness to ATRA. Complementing our work with the p50-RARA transgenic mice, retroviral co-transduction of normal bone marrow with βcV449E plus either p50-RARA or PML-RARA generated APL-like myeloid leukemias. Although retrovirally generated βc/p50-RARα and βc/PML-RARα leukemias were nearly identical, the PML fusion appeared associated with a subtle decrease in differentiation relative to the p50 fusion. In contrast to these X-RARα fusions, preliminary experiments suggest that RCRα (a homodimeric form of RARα in which the dimerization interface of RARα has been replaced by the dimerization domain of the homodimeric COUP-TF transcription factor) does not readily cooperate with βcV449E to cause leukemia. This finding lends support to the hypothesis that the decreased binding site selectivity of X-RARα fusions contributes to APL pathogenesis. Furthermore, we have generated a novel X-RARα fusion, F3-RARα, in which three copies of the F36M mutant of the Rapamycin-binding protein have been fused to RARα. This protein mimics the in vitro effects of PML-RARα on transcription & differentiation, and de-dimerization of F3-RARα by rapamycin reverses these effects. Additional experiments in vivo with F3-RARα are expected to further confirm the importance of abnormal transcription factor dimerization in leukemia pathogenesis.
Pim Kinases Mediate Viability Signals Downstream of the Tyrosine Kinase Oncogenes BCR-ABL and FLT3-ITD.
Authors: Sattler Martin , Babendreier Emily , Chu Stephanie C. , Gramlich Jessica L. , Podar Klaus , Griffin James D.
Published: April 2020

Pim1 and Pim2 belong to a family of serine/threonine kinases that are overexpressed in many leukemias. Pim1 has previously been shown to cooperate with the v-myc oncogene to transform hematopoietic cells with murine transforming viruses and overexpression of Pim2 has been shown to induce growth factor independence in a factor dependent murine BaF3 cell line. Protein expression of both Pim1 and Pim2 is low or absent in non-transformed hematopoietic cells but was found to be rapidly increased upon transformation by BCR-ABL or FLT3 with an internal tandem duplication (ITD). The exact contribution of the Pim kinases to transformation in these cells is unknown. BaF3 cell lines were created that stably expressed either BCR-ABL or FLT3-ITD tyrosine kinases, and in which either Pim1 or Pim2 could be induced with doxycycline. Treatment of BCR-ABL or FLT3-ITD expressing cells with small molecule kinase inhibitors specific for either ABL or FLT3 led to inhibition of cell growth, increased apoptosis, and downregulation of Pim expression as expected. However, if Pim2, and to a lesser extent, Pim1 expression was maintained by doxycycline, there was a substantial increase in both viability and cell growth. The molecular mechanisms by which Pim proteins exhibit their effects on target cells are not known. Using parental growth factor dependent BaF3 cell lines with doxycycline inducible Pim1 and Pim2, we show that expression of either of the Pim proteins is sufficient, by itself, to reduce apoptosis and induce modest cell growth. The effects of Pim overexpression on several pathways known to be associated with viability were studied. We found that while Pim over-expression does not activate Akt, it does result in the phosphorylation of a known Akt target, GSK-3β, a regulator of cell cycle progression by targeting the stability of cyclin D proteins. This suggests that Pim proteins may mediate their biological effects through regulation of components in the phosphatidylinositol-3′ kinase signaling cascade, independent of Akt activation. These results further suggest that upregulation of Pim kinases significantly contributes to transformation. Inhibition of Pim kinases could have beneficial therapeutic effects in leukemias.
A Cell-Based Screening Method for Resistance of Bcr-Abl Positive Leukemia Identifies the Mutation Pattern for an Alternative Abl Kinase Inhibitor.
Authors: von Bubnoff Nikolas , Veach Darren R. , van der Kuip Heiko , Aulitzky Walter E. , Saenger Jana , Seipel Petra , Bornmann William G. , Peschel Christian , Clarkson Bayard , Duyster Justus
Published: April 2020

The increasing impact of targeted cancer treatment demands strategies to identify and evaluate resistance mechanisms toward kinase inhibitors prior to their therapeutic application. Point mutations within the Bcr-Abl kinase domain constitute the major mechanism of resistance toward imatinib mesylate in Philadelphia-positive (Ph+) leukemia. Using Bcr-Abl-transformed Ba/F3 cells, we established a cell-based screening strategy for the prediction of specific kinase mutations that cause resistance toward kinase inhibitors. With imatinib at clinically relevant concentrations, we generated 368 resistant Ba/F3 sublines that were derived from resistant colonies. Thirty-two different single point mutations within the kinase domain of Bcr-Abl were identified in twenty-five per cent (liquid culture conditions) and seventy-two per cent (solid culture conditions) of these lines at known and novel positions. Using imatinib, the pattern and relative frequency of mutations reflected matters observed in patients with imatinib resistance. We then applied this screen to the pyrido-pyrimidine PD166326 (PD16), an investigational Abl kinase inihibitor. Compared to imatinib, we observed a five to seven times lower frequency of resistant colonies with equipotent concentrations of PD16. In addition, PD16 produced a distinct pattern of Bcr-Abl mutations. P-loop, A-loop and the known imatinib contact site T315 were affected with both inhibitors, whereas C-helix and SH2 contact sites were affected in imatinib resistant colonies exclusively. In contrast to imatinib, where kinase domain mutations were still widely distributed over the kinase domain even at at 4μM, mutations observed with PD16 at a concentration of 100nM narrowed to the exchange at position T315 to iseulicine. We did not detect mutations outside the kinase domain. Some resistant sublines displayed increased Bcr-Abl activity. Mutations that were derived from the screen were cloned and examined for the extent of cross-resistance to both inhibitors. The majority of mutations were effectively suppressed by PD16 at 50–500nM. In contrast, only few mutations were inhibited by imatinib at 5–10μM. However, exchanges at position F317 mediated resistance toward PD16, but were inhibited by standard concentrations of imatinib. Since this cell-based system produced results that are clinically significant, it may be used to predict resistance mutations in Bcr-Abl and other oncogenic kinases like cKit, EGFR, FIP1L1-PDGFRalpha or FLT3 towards clinically applicated and investigational drugs. Thus, this robust and simple screening strategy provides a rational basis for combinatorial and sequential treatment strategies.
Prognostic Impact of Minimal Residual Disease (MRD) in Children Is Different in B or T Lineage Acute Lymphoblastic Leukemia: Results of Trial AIEOP-BFM ALL 2000.
Authors: Schrappe Martin , Rizzari Carmelo , Mann Georg , Möricke Anja , Valsecchi Maria Grazia , Zimmermann Martin , Bartram Claus R. , Panzer-Grümayer Renate , Schrauder André , Cazzaniga Gianni , Flohr Thomas , Parasole Rosanna , Reiter Alfred , Ludwig Wolf-Dieter , Nigro Luca Lo , Basso Giuseppe , De Rossi Giulio , Barisone Elena , Niggli Felix , Gadner Helmut , Conter Valentino
Published: April 2020

Slow early response indicates poor prognosis in childhood ALL. We aimed to evaluate if post-induction MRD levels had different prognostic impact in precursor B-cell (pB) or T-cell ALL. From 07/2000 to 06/2006, 4730 pts with ALL were enrolled in trial AIEOP-BFM ALL 2000. MRD levels were centrally measured by real-time quantitative polymerase chain reaction using the identification of clone-specific T-cell receptor and immunoglobulin gene rearrangements. MRD study time-points (TP) were treatment day 33 (TP1, end of induction) and day 78 (TP2, after consolidation). To define MRD negativity, two markers with a sensitivity of at least 10−4 were required. Patients were treated with BFM induction (protocol I-A), consolidation (I-B), extra-compartment/intensified consolidation (HD-MTX in non-high-risk patients, pulses in high-risk patients), reinduction, and maintenance. MRD analysis at one or two time points suceeded in 3707 pts; the immunophenotype was available from 3636 pts. MRD levels and corresponding estimated 5-year event-free survival (5y-pEFS) comparing pB- and T-ALL are shown in Table 1 (3yrs median follow-up). MRD response in T-ALL was slower than in pB-ALL resulting in a higher percentage of pts with high MRD load in T-ALL. In pB-ALL as well as T-ALL, high MRD levels at TP2 were well predictive to identify pts with poor prognosis. For prediction of good prognostic subgroups, TP1 was more appropriate identifying a subgroup with excellent 5y-pEFS of >90% in case of MRD negativity. Specificity of TP1 was poor in T-ALL if the pB-ALL criteria of MRD negativity were applied. If MRD low positive and MRD negative T-ALL pts were combined, the discrimination was as good as in pB-ALL. The optimal choice of MRD evaluation time points depends on biological factors and treatment, and is most relevant for MRD-based risk stratification.

Table 1 pB-ALL T-ALL n % 5y-pEFS % (SE) n % 5y-pEFS % (SE) all 3177 100% 82.3 (1.0) 459 100% 77.2 (2.2) MRD TP1     neg 1399 44.1 92.5 (1.0) 75 16.4 94.3 (2.8)     10E-4/−5 1122 35.4 81.9 (1.7) 116 25.4 91.2 (2.8)     10E-3 393 12.4 66.4 (3.5) 110 24.1 75.3 (4.6)     ≥10E-2 256 8.1 53.2 (4.3) 156 34.1 59.8 (4.5) MRD TP2     neg 2464 77.6 87.7 (1.0) 220 47.9 91.9 (2.0)     10E-4/−5 523 16.5 68.9 (2.9) 143 31.2 76.6 (3.9)     10E-3 107 3.4 56.3 (6.5) 58 12.6 50.2 (8.1)     ≥10E-2 82 2.6 38.0 (7.3) 38 8.3 33.2 (8.3)
Evaluation of the Spectrin Compartment Size in Normal and Diseased Red Blood Cells by Single Particle Tracking.
Authors: Kodippili Gayani C. , Spector Jeff , Sullivan Caitlin , Labotka Richard , Ritchie Ken , Low Philip S.
Published: April 2020

In a process termed “hop diffusion”, membrane proteins diffuse randomly within a cytoskeletally-enclosed compartment over short time scales, periodically hopping from one compartment to the next. Band 3 diffuses in the erythrocyte membrane bilayer in a manner that is thought to be restricted by the size and stability of the spectrin network. Although a fraction of band 3 may exhibit reduced mobility due to its association with spectrin (mediated by proteins such as ankyrin and adducin), another subpopulation of band 3 is thought to diffuse freely within the spectrin compartments, hopping from one corral to the next by transient opening of the barrier (presumably a spectrin tetramer) separating adjacent corrals. Based on this hypothesis, measurement of the compartment size and hopping frequency of a band 3 molecule in the erythrocyte membrane can provide information on the structure and stability of the spectrin network. We have combined single particle tracking (SPT) techniques with high-speed video microscopy to determine the average compartment size and diffusion coefficient of the mobile fraction of band 3 in both normal and diseased red blood cells (RBCs). Streptavidin-linked quantum dots (525nm emission) and 40 nm fluorescent beads were attached to band 3 via a DIDS-biotin-linker, and fluorescence microscopy coupled to a high speed camera was used to determine the position of band 3 with a time resolution of 8 ms (120 fps). Labeling specificity was demonstrated by blotting with streptavidin-horseradish peroxidase, and flow cytometry studies established the binding constant of DIDS-biotin for band 3 in the intact cell of ∼1-2*10−5 M. For diffusion studies, RBCs were incubated with 10−11 M conjugate in order to attach only one or two streptavidin-quantum dots per cell. SPT data were collected on both healthy and pathologic human RBCs, including HbSS (sickle cells), HbSC (sickle cell hemoglobin C), HbSBo (sickle cell zero-beta-thalasamia), HbSB+ (sickle cell beta-plus-thalasamia), HS (hereditary spherocytosis), and HPP (hereditary pyropoikilocytosis). Movement within the observed compartments was found to be random, with an average diagonal length for the mobile fraction in healthy cells of ∼100nm and a diffusion coefficient of ∼1–2*10−10cm2/s. HbSS cells, however, displayed a comparatively smaller compartment size (∼60–80nm) with a lower diffusion coefficient (0.1−1*10−10cm2/s). In contrast, HPP cells exhibited an average compartment size of ∼133nm with a diffusion coefficient of 6*10−10cm2/s. SPT data further suggest the presence of multiple populations of band 3, exhibiting both free diffusion and restricted mobilities with different characteristics. Details of band 3 diffusion in all of the aforementioned diseased and normal blood cells will be provided, and a model accounting for the multiple populations of band 3 will be presented.
Prognostic Role of Glutathione S-Transferase (GST) Polymorphisms in Acute Myeloid Leukemia.
Authors: Voso Maria Teresa , Hohaus Stefan , Guidi Francesco , Fabiani Emiliano , D’Alo’ Francesco , Groener Silia , Spaeth Daniela , Dohner Konstanze , Leone Giuseppe , Dohner Hartmut , Schlenk Richard F.
Published: April 2020

Glutathione S-transferases (GST) are phase II detoxification enzymes, involved in the metabolism of carcinogens and anti-cancer drugs. Since GSTs have been also shown to interact with kinase complexes during oxidative or chemical stress-induced apoptosis, their polymorphic variants may account for differences in outcome following chemotherapy between individuals. Studying 106 patients (pts) with acute myeloid leukaemia (AML), we have shown that individuals with a GSTM1 and/or GSTT1 deletion were at higher risk of relapse and of shorter overall survival (Voso et al, Blood100:2703, 2002). The aim of the present study was to confirm or previous findings and to better define the role of GSTM1 and GSTT1 deletions in the context of a molecularly well-characterized cohort of AML-patients who were treated within two prospective clinical trials of the German-Austrian Acute Myeloid Leukemia Study Group (AMLSG) In addition to polymorphisms in GSTM1 and GSTT1, we were also interested in the role of the GSTP1(Ile105Val) polymorphism on outcome. Based on our previous work, we intended to show a 20% difference in the 3-year survival rate. Therefore the estimated power for our current study including 350 patients to confirm the previous findings was 98%. We studied 354 patients (pts) with AML, treated according to two AMLSG prospective multicenter clinical trials (HD98-A for pts with age < 60 years, 254 pts; HD98-B for pts > 60 yrs, 100 pts). The Ile105Val polymorphism of the GSTP1 gene (GSTP1*105) was analyzed by PCR-RFLP, while GSTM1 and GSTT1 homozygous deletions (GSTM1*0 and GSTT1*0) were studied using a multiplex PCR technique, including the housekeeping gene BCL-2 as internal control. GST genotypes were not associated to patients’ characteristics, including type of AML, cytogenetic risk group, presence of FLT3-ITD and abnormal laboratory parameters, and response to induction chemotherapy. GSTP1*105Val was associated with a significant better relapse-free survival using age-stratified test (adjusted HR, 0.7; 95% C.I., 0.51–0.98), while GSTT1 and GSTM1 genotypes had no impact on RFS. A Cox proportional hazard model on RFS including GST genotypes, LDH level, cytogenetic risk group, and FLT3-ITD mutation status showed that the GSTP1 genotype was a significant variable associated with a favourable outcome (n= 236 pts, HR, 0.62; 95% C.I., 0.44–0.88). Limiting the analysis to patients with a normal karyotype, the GSTP1 105Val allele was associated in the multivariable model on RFS with a 51% risk reduction (HR, 0.49; 95%C.I., 0.30–0.81). In conclusion, GSTP1*105Val seems to be of prognostic value, whereas the prognostic value of GSTM1 and GSTT1 reported in previously could not be confirmed.
Use of Post-Treatment Clinical Data To Predict Response to Decitabine.
Authors: Estey Elihu , Issa J.-P. , Garcia-Manero Guillermo , Faderl Stefan , Kantarjian Hagop
Published: April 2020

Best response of AML or MDS to decitabine (DAC) may require > 3 courses. Identification of pts whose response to initial courses suggests they are unlikely to respond further to subsequent courses would allow such pts to receive other therapies more expeditiously. 104 pts, median age 69, received DAC (20 mg/m2 IV daily × 5 days every 5 weeks) for newly- diagnosed MDS (80, 76 IPSS int-2 or high) or AML (24) from 9/00-5/07. After 3 courses, 33 of the 104 were in CR, 6 were dead, and 32 had been removed from study, with the remaining 32 receiving more DAC. The probability of CR decreased to < 10% on all courses after the 5th:

Course# Pts Receiving CR Deaths/Off Study Received Next Course after course Course 1 104 7 (7%) 2/15 80 2 80 16 (20%) 4/12 48 3 48 10 (21%) 0/6 32 4 32 5 (16%) 0/5 22 5 22 3 (14%) 0/3 16 6 16 1 (6%) 1/3 11 7–15 11 1 (9%) 1/9 0

Results were qualitatively similar for AML and MDS. We divided the 32 pts who received ≥ 4 courses according to whether after course 3 their:

marrow showed a response, defined as a p < 0.05 difference between the pre-Rx and post course 3 blast count, platelet count increased > 30, 000 if < 100,000 pre-Rx, or neutrophil count increased > 500 if < 500 pre Rx; criteria 2 and 3 were adapted from those of the IWG. 8/14 pts with vs. 1/11 without a marrow response after course 3 entered CR on subsequent courses (p = .02, 6/10 vs. 1/10 considering only MDS).

The corresponding figure for platelet response/no response was 5/8 vs. 2/11 (3/5 vs. 2/9 for MDS). The median number of courses after the 3rd given pts with marrow response was 2 (range 1–10) vs. 3 (range 1–12) for pts without marrow response. Our data suggest that if CR is the goal, pts should not receive more than 5 courses of DAC and no more than 3 if marrow response has not occurred after the 3rd.
The Bone Resorption Marker Serum-ICTP and the Circulating Proteasome Levels Separate ISS-Stages 1–3 and Are the Most Powerful Prognostic Factors for Overall Survival in Newly Diagnosed Symptomatic Multiple Myeloma.
Authors: Jakob Christian , Liebisch Peter , Egerer Karl , Sterz Jan , Kaiser Martin , Rötzer Susanne , Heider Ulrike , Rademacher Jessica , Fleissner Claudia , Kloetzel Peter M. , Terpos Evangelos , Sezer Orhan
Published: April 2020

Prognostic markers are important to identify high-risk patients in Multiple Myeloma (MM). Several prognostic models have been published, including parameters of tumor burden and cytogenetics. Recently the International Staging System (ISS) for Multiple Myeloma, which includes beta2-microglobulin (beta2-MG) and albumin, has been introduced for newly diagnosed patients with symptomatic MM. We previously reported about the prognostic significance of circulating proteasome levels (cProteasomes) in MM patients (Jakob et al., Blood 2007). In the present study we investigated the prognostic relevance of the bone resorption marker carboxy-terminal telopeptide of type-I collagen (ICTP) and cProteasome levels in comparison to the classical prognostic factors (beta2-MG), albumin, deletion 13q14 and type of chemotherapy (high-dose-therapy versus conventional dose chemotherapy) in 92 patients with newly diagnosed active MM. ICTP and cProteasome were significantly elevated parallel to ISS stages (P<0.001). ICTP (cut off: normal value), cProteasome levels (cut off: median value) and a combined ICTP-cProteasome score (1: ICTP < normal value and cProteasome < median; 2: one of both parameters elevated; 3: both parameters elevated) were significant univariate prognostic factors for overall survival in active MM (P<0.001, P=0.002 and P<0.001, respectively). Survival rates at 5-yrs were 95%, 66% and 27% in the groups of patients with ICTP-cProteasome score 1, 2 and 3, respectively. In a further analysis ICTP alone and the combined ICTP-cProteasome score significantly separated each of the three ISS stages into two distinct groups with a better versus worse prognosis. In a multivariate Cox regression analysis, including beta2-MG, albumin, deletion 13q14, type of chemotherapy, cProteasome levels and ICTP, ICTP was the parameter with the strongest prognostic power (P<0.001, hazard-ratio: 7.9) and cProteasomes (P=0.011, hazard-ratio: 3). Our study underlines that the activity of myeloma bone disease, as reflected by the collagen-I degradation product ICTP, has a major impact on the prognosis of symptomatic MM. This result is in line with published data showing a positive feedback between myeloma cells and osteoclasts, i.e. a vicious cycle. The inclusion of the bone resorption marker ICTP and cProteasome levels into multivariate models add substantial prognostic information on overall survival in newly diagnosed active MM.
Multiple Myeloma (MM) Is Characterized by Genomic Instability Regardless of Ploidy Categories and Degree of Karyotypic Complexity Is an Important Prognostic Factor.
Authors: Chng Wee J. , Keats Jonathan J. , Braggio Esteben , Baker Angela , Bergsagel P. Leif , Carpten John , Fonseca Rafael
Published: April 2020

MM is characterized by widespread genomic instability. Two broad genetic subtypes of MM defined by ploidy exist. Hyperdiploid MM (H-MM) is characterized by trisomies of chromosome 3, 5, 7, 9, 11, 15, and 19, and generally lacked primary translocations involving the immunoglobulin heavy chain locus (IgH) whereas the non-hyperdiploid MM (NH-MM) is characterized by primary IgH translocations such as t(11;14), t(4;14) and t(14;16). Using high-density array comparative genomic hybridization (aCGH, Agilent 44K array), we catalogued the different aberrations on each chromosome arm and also the total number of aberrations per tumor (NAPT), as an indication of the degree of genomic instability, in a large cohort of 194 MM patients. Both H-MM and NH-MM have high number of genomic aberrations, 15.8±7.5 (mean ± standard deviation) versus 20.6±17.0 respective. NH-MM has significantly more genomic losses (11.8±11.0 versus 4.1±3.8), whereas H-MM has slightly more genomic gains (11.7±5.0 versus 8.8±9.0). Most of the abnormalities in NH-MM are structural abnormalities whereas they are mainly whole chromosome abnormalities in H-MM. Based on these abnormalities, we cluster the patients using hierarchical clustering. The 2 main branch of the resulting dendogram delineate hyperdiploid and non-hyperdiploid patients. Sub-clusters of each ploidy subtype are apparent. For H-MM, a cluster with mainly trisomies, including that of chromosome 21, and another cluster with additional deletions (13, 16q, and less trisomy 21 and trisomy 11) were apparent. For NH-MM, a group with 1q amplification and 13 deletion, which also tend to have loss of chromosome 4, 6q, 16q and 14, and another group with little abnormalities or just 6q loss, and chromosome 13 and 14 loss were identified. We identified some genetic difference between NH-MM and H-MM in additional to known difference such as the trisomies and chromosome 13 deletion. Loss of chromosome 4 (18% versus 0.9%, p<0.0001), loss of chromosome 14 (33% versus 16%, p=0.02) and loss of chromosome 22 (21% versus 4%, p=0.0008) are more common in NH-MM. Based on the distribution of NAPT, a cut-off of 20 abnormalities per tumor segregate the patients into 2 groups with significantly different survival. Those with greater NAPT have significantly shorter survival (median overall survival of 20 months versus 88 months, Log-rank p=0.006). There is no correlation between NAPT and ploidy or presence of chromosome 1 abnormalities suggesting that genomic complexity itself is associated with survival. Our study showed that both H-MM and NH-MM are genomically unstable although the pattern of abnormalities is distinct. In addition, based on clustering analysis, new pattern of genetic abnormalities are observed that allow further refinement of the ploidy subtypes. In addition, genomic complexity is also an important prognostic factor.