bluuebunny/crossref_metadata_2025 huggingface dataset:
Postremission Therapy with an Allogeneic Transplantation from an HLA-Matched Family Donor Seems To Overcome the Negative Prognostic Impact of FLT3-ITD in Younger Patients with Acute Myeloid Leukemia Exhibiting a Normal Karyotype.
Authors: Schlenk Richard F. , Krauter Jürgen , Fröhling Stefan , Späth Daniela , Schäfer Irina , Hein Stefan , Döhner Hartmut , Ganser Arnold , Döhner Konstanze ,   for the German-Austrian AML Study Group (AMLSG)
Published: April 2020

Internal tandem duplications (ITD) of the FLT3 gene have been identified as negative prognostic marker in patients with acute myeloid leukemia (AML) exhibiting a normal karyotype. To evaluate the impact of different postremission strategies, such as high-dose cytarabine based chemotherapy (chemo), autologous (auto-SCT) or allogeneic stem cell transplantation (allo-SCT), in patients with normal karyotype and FLT3-ITD, we initiated a pooled data analysis within the prospective treatment trials AML-2/95, AML-1/99, AMLHD93, and AMLHD98A.

Methods: All patients (age 16–60 years) received two cycles of induction therapy with standard-dose cytarabine combined with etoposide and idarubicin. After a first consolidation therapy, patients were assigned to allo-SCT if an HLA-identical sibling donor was available in all four trials. In the AML-2/95 and AMLHD93 trials, all other patients were assigned to chemo, whereas in the AML-1/99 and AMLHD98A trials patients were randomized between chemo and auto-SCT. Patients were analyzed for the presence of FLT3-ITD by genomic DNA PCR and conventional gel electrophoresis. All statistical tests were stratified for the variable “study group”.

Results: Between 1993 and 2004 872 patients exhibiting a normal karyotype were registered. To date, results of FLT3 mutation analysis are available for 620 patients, and 182 (29%) exhibited an ITD. Response to induction therapy was 72% and 79% in the FLT3-ITD positive group and the FLT3-ITD negative group, respectively. After a median follow-up of 41 months, relapse-free (RFS) and overall survival (OS) were 33% versus 51% (p<0.0001) and 27% versus 46% (p<0.0001) in the FLT3-ITD positive and the FLT3-ITD negative groups, respectively. Analysis based on the availability of an HLA-matched family donor revealed a significantly better RFS (p=0.007) of 60% for patients with a donor (n=26) versus 21% for patients without a donor (n=102).

Conclusion: In AML patients with normal karyotype exhibiting a FLT3-ITD mutation who achieve CR after induction therapy, consolidation with allo-SCT seems to overcome the negative impact of FLT3-ITD on outcome.
Prognostic Impact of Anti-Apoptotic and Multidrug Resistance Phenotypes in Elderly Acute Myeloid Leukemia Patients.
Authors: Vidriales María-Belén , Suarez Lilia E. , García-Laraña Jose , García-Boyero Raimundo , Moreno María-José , Bernal Teresa , Reina Vicencia Martín , González-San Miguel Jose D. , Pérez José J. , Fernández-Abellán Pascual , García-Suarez Julio , San Miguel Jesús F. , Orfao Alberto ,   for the Pethema Cooperative Group
Published: April 2020

Elderly patients with acute myeloid leukemia (AML) have an unfavorable outcome, which has been related to both the poor performance status of many of these patients and the biological characteristics of the malignant clone, including the expression of multidrug resistance (MDR) phenotypes. We have quantitatively analyzed by flow cytometry the expression of apoptosis- (bcl-2, bax, APO2.7) and MDR- (P-gp, MRP, LRP) associated proteins in a group of 117 elderly (>65 years) uniformly treated de novo AML patients according to the Pethema LMA-98 protocol. Upon analyzing factors influencing the response to induction therapy, as expected, patients with good and intermediate cytogenetics (n=76) more frequently achieved morphological complete remission (mCR) (63%) than patients having poor cytogenetics (n=23) (mCR: 30%). In addition, CD34 expression also influenced response; thus, 81% (21/26) of CD34-negative patients achieved mCR vs only 46% (42/91) of CD34-positive cases (p=0,008). Neither age nor WBC counts showed a significant influence in response rate. As far as apoptosis and MRD proteins is concerned, interestingly, responding cases showed a lower expression of the bcl-2, MRP and LRP proteins (bcl-2 RFI (relative fluorescence intensity) 10±5.5 vs 13±6, p=0.02; MRP RFI 1.9±0.8 vs 2.7±1.8, p=0.008; LRP RFI 6.2±4.9 vs 8.7±7.5, p=0.01 in responding vs non-responding patients). By contrast, expression of APO 2.7, Bax, and MDR-1 did not influenced response. Analysis of relapse free survival showed that only the number of cycles of chemotherapy required to achieve mCR had prognostic influence (p=0.008), with no significant influence for age, WBC counts, CD34 expression, or cytogenetics. In turn, a high percentage of early apoptotic cells in bone marrow at diagnosis (p=0.01), as well as a low bcl-2/bax ratio (p=0.05), and low MRP expression (p=0.04) were associated with a prolonged RFS. Moreover, upon grouping AML patients according to the expression of MRP and the bcl-2/bax ratio (> and < of the mean of both parameters), only patients with both low MRP expression and low bcl-2/bax ratio (n=16) achieved a plateau phase in the RFS curve after 20 months of follow-up, while the remaining patients showed a continuous relapse trend.

In summary, our results show that, in addition to high bcl-2 and bcl-2/bax ratio, a high expression of the LRP and MRP multidrug resistant proteins have an adverse prognostic influence in elderly AML patients. The combination of these parameters contribute to identify distinct groups of patients at a different risk of relapse.
Clinical Characteristics and Prognostic Implications of NPM1 Mutations in Acute Myeloid Leukemia.
Authors: Suzuki Tatsuya , Kiyoi Hitoshi , Ozeki Kazutaka , Tomita Akihiro , Suzuki Ritsuro , Kodera Yoshihisa , Miyawaki Shuichi , Asou Norio , Kuriyama Kazutaka , Yagasaki Fumiharu , Shimazaki Chihiro , Akiyama Hideki , Nishimura Miki , Motoji Toshiko , Shinagawa Katsuji , Takeshita Akihiro , Ueda Ryuzo , Naoe Tomoki
Published: April 2020

Nucleophosmin (NPM) is a nucleolar protein with multi-functions including centromere duplication, nuclear-cytoplasmic shuttling, ribosomal biogenesis, p53 stability. NPM1 mutations were found in a large number of patients with acute myeloid leukemia (AML) especially with normal karyotype. The mutations lead to the aberrant subcellular localization of NPM protein. However, their impacts on clinical outcome remain controversial. We screened the mutations of NPM1 in 257 AML patients and analyzed the clinical significance. NPM1 mutations were found in 64 of 257 patients (24.9%). Seven types of mutations, including four novel mutations, were identified. NPM1 mutations were associated with normal karyotype, FLT3 mutations (both FLT3-ITD and D835 mutation) but not with other gene alterations such as N-RAS, p53 mutations and partial tandem duplication of the MLL gene. In 190 patients except the M3 subgroup, who were treated according to the protocol of Japan Adult Leukemia Study Group, the multivariate analysis revealed that NPM1 mutation was a favorable factor for achieving complete remission, but significantly associated with relapse. A sequential analysis, using paired samples obtained at diagnosis and relapse in 39 patients, revealed that NPM1 mutations were lost at relapse in 2 of the seventeen patients who had NPM1 mutations at diagnosis and none of the patients, who did not have NPM1 mutations at diagnosis, gained NPM1 mutations at relapse.

Our results suggest that NPM-mutated AML should be a distinct subgroup with specific clinical characteristics and outcome. Loss of NPM mutations at relapse implies that NPM mutation is not necessarily a primary genetic alteration and that these leukemic clones could be sensitive to chemotherapy.
Multivariate Analysis of Prognostic Impact of Flow Cytometrically Determined Minimal Residual Disease in Patients with Acute Myeloid Leukemia.
Authors: Kern Wolfgang , Voskova Daniela , Schoch Claudia , Schnittger Susanne , Haferlach Torsten
Published: April 2020

Treatment of acute myeloid leukemia (AML) is adapted according to individual risk profiles. The assessment of levels of minimal residual disease (MRD) is feasible in the vast majority of patients with AML and may significantly improve prognostication. We applied multiparameter flow cytometry to highly sensitively quantify MRD in patients with AML and analyzed its prognostic impact in a multivariate analysis. A total of 356 patients receiving standardized intensive antileukemic treatment was analyzed at different checkpoints: CP1 (up to day 28, n=156), CP2 (day 28–60, n=122), CP3 (day 61–120, n=195), CP4 (day 121–365, n=172), and CP5 (after day 365, n=73). Leukemic cells were identified by their individually defined leukemia-associated aberrant immunophenotypes (LAIPs). MRD levels were calculated as the logarithmic difference (LD) between LAIP-positive cells at diagnosis and LAIP-positive cells at follow-up assessment. The median LD amounted to 2.18 (range, −0.14 to 4.20) at CP1, 2.31 (−0.03 to 4.17) at CP2, 2.49 (0.11 to 4.17) at CP3, 2.58 (−0.28 to 4.28) at CP4, and 2.87 (0.46 to 4.02) at CP5. A higher LD (continuous variable) was related to a better event-free survival (EFS; CP1, p=0.0002; CP2, p=0.00001; CP3, p=0.0002; CP4, p<0.00001; CP5, p=0.00007) and to a better overall survival (OS; CP1, p=0.004; CP2, p=0.001; CP3, p=0.021; CP4, p=0.00006). Other parameters related to EFS and OS were age and cytogenetics in the present series. The prognostic impact of MRD levels on outcome was idependent of cytogenetics and age for EFS (CP2 to CP5) and OS (CP2 and CP4). MRD was the most important prognostic parameter at CP4 and CP5. Separation of patients into two groups, respectively, by the median LD resulted in significant differences in EFS at all CPs and in OS at CP1 to CP4. The largest difference was observed at CP4: median EFS, 57.1 vs. 13.7 months, p<0.00001; 3-year-OS, 95% vs. 65%, p=0.0003. Determination of MRD levels by multiparameter flow cytometry provides a highly powerful and independent prognostic parameter which is applicable to the total of an AML population. It should be evaluated as a stratification parameter in clinical trials.
Murine Ultrasonography Confirms That Rapamycin Is Effective Against Autoimmune Lymphoproliferative Syndrome (ALPS).
Authors: Teachey David T. , Axsom Kelly , Brown Valerie I. , Obzut Dana , Grupp Stephan A.
Published: April 2020

Autoimmune Lymphoproliferative Syndrome (ALPS) is a disorder of abnormal lymphocyte survival caused by defective Fas-mediated apoptosis. Patients with ALPS develop lymphadenopathy, hepatosplenomegaly, and an increased number of an unusual T cell population, characterized as T cells that are CD3+ CD4− CD8− and referred to as double negative T cells (DNTs). Although ALPS is a rare disorder, we have shown it may be more common than previously appreciated. Treatment options for patients with ALPS are limited. Rapamycin, an mTOR inhibitor, has been shown to induce apoptosis in nonmalignant B and T lymphocytes and we have demonstrated its potential efficacy in models of lymphoid leukemia. Because ALPS is caused by defective lymphocyte apoptosis, we hypothesized rapamycin would be effective in ALPS by inducing apoptosis in these abnormal cells, controlling the lymphoproliferation that is the hallmark of the disease. We confirmed this hypothesis by using a murine model of ALPS, CBA-lprcg, treating animals that had developed significant lymphadenopathy and splenomegaly with rapamycin. We followed response to treatment with serial assessment of DNTs in peripheral blood and lymphoid tissue by flow cytometric determination and by serial monitoring of lymph node and spleen size with small animal ultrasound. This is the first report to use this novel and powerful tool to measure murine lymphoproliferation and we found 3-dimensional ultrasound measurements in the mice correlated to actual tissue measurements at sacrifice (r = 0.9648, p = 0.0001). We found a dramatic and statistically significant decrease in DNTs (p = 0.02), lymphadenopathy (p = 0.003), and splenomegaly (p = 0.03) after only 4 weeks when comparing mice treated with rapamycin to control animals (Table). We found no toxicities and no evidence of myelosuppression in treated animals. We performed immunoblots to evaluate a major mTOR signaling pathway intermediate (phospho-S6) on lymph node biopsies obtained from mice before and after treatment with rapamycin and found a correlation between clinical response and biochemical response (downregulation of phospho-S6). In summary, rapamycin, a safe and well tolerated medicine, is effective in murine ALPS and its use should be explored in patients with the disease.

Rapamycin Effective in ALPS Disease Parameter (1) Rapamycin Treated(2) Vehicle Control(2) p value (1) Average number of DNTs, volume of lymph nodes, and area of spleens were the same between groups at initiation of treatment. (2) Average (range) after 4 weeks of treatment DNTs of blood by FACS (mm3) 2440 (1343 – 3756) 9957 (4426 – 20384) 0.02 Lymph node volume by ultrasound (mm3) 52 (3 – 193) 760 (366 – 1043) 0.003 Splenic area by ultrasound (mm2) 54 (20 – 103) 179 (95 – 336) 0.03
Bortezomib Alters Peripheral Blood Lymphocyte Subsets in Patients with Multiple Myeloma.
Authors: Uy Geoffrey L. , Holt Matthew S. , Fisher Nicholas M. , Devine Steven M. , Tomasson Michael H. , DiPersio John F. , Vij Ravi
Published: April 2020

Bortezomib (VELCADE®) is a potent inhibitor of the proteasome which exerts its antimyeloma effect in part by blocking the activation of NF-κB. As NF-κB is critical for lymphocyte development and survival, there is great interest in harnessing the potential immunomodulatory effects of bortezomib. In murine hematopoietic transplantation models, bortezomib inhibits in vitro mixed lymphocyte responses and promotes the apoptosis of alloreactive T cells protecting against acute graft-versus-host disease. However, no data exists on the in vivo effects of bortezomib on human T cells. To characterize the effects of bortezomib on immune function, we profiled peripheral blood lymphocytes subsets and T cell associated cytokines in 39 patients with multiple myeloma. Two cycles of bortezomib 1.3 mg/m2 were administered by intravenous infusion on days 1, 4, 8, and 11 of a 21-day treatment cycle. The patients had received prior induction chemotherapy and would proceed to autologous transplant following treatment with bortezomib. Study population consists of 23 male and 16 female patients with the median age of 56 years (range 38–69). Myeloma characteristics at diagnosis were as follows (number of patients): IgG (28), IgA (10), light chain only (1), with stage I (1), II (12), or stage III (26) disease. Peripheral blood was collected at baseline (cycle 1, day 1) and at one week after the last dose of bortezomib (cycle 2, day 18) and analyzed for lymphocyte subsets by standard multicolor flow cytometry. Th1 and Th2 serum cytokines were measured at the same timepoints using a multiplexed cytometric bead array (BD Biosciences). Following treatment with bortezomib, no significant changes were detected in either Th1 or Th2 serum cytokine levels: IL-2 (p=0.116), TNF-alpha (p=0.854), IFN-gamma (p=0.070), IL-4 (p=0.240), IL-6 (0.236), IL-10 (0.151) as analyzed by Wilcoxon signed ranks test. Analysis of lymphocyte subsets using a paired student’s T-test demonstrated a 38% decrease in CD56+ NK cells (p=0.02) and a 26% increase in CD4/CD8 ratio (p=0.0006) which appears to be secondary to a decrease in CD8+ cytotoxic T-cells (p=0.054). (Table 1.) In conclusion, we observe an alteration of lymphocyte subsets following only two cycles of bortezomib. Further analysis of the effects of long term treatment with bortezomib is warranted. These studies may provide insights into the role of bortezomib as an immunomodulatory agent.

Peripheral Blood Lymphocyte Subsets Pre-bortezomib (/mm 3 ) Post-bortezomib(/mm 3 ) Difference(/mm 3 ) P-value CD2 1446 1259 −187 0.085 CD3 1273 1160 −113 0.28 CD4 842 802 −40 0.54 CD8 412 337 −75 0.055 CD19 90 94 4 0.86 CD20 87 95 8 0.78 CD56 206 148 −58 0.022 CD4/CD8 ratio 2.53 3.19 0.66 0.0006
Trafficking and Expansion of NK Cells in Murine Models of Bone Marrow Transplantation.
Authors: Olson Janelle A. , Negrin Robert
Published: April 2020

Allogeneic hematopoietic cell transplantation has proven to be an effective treatment for hematologic malignancies and some solid tumors. However, the high incidence of graft versus host disease (GVHD) as a complication of this treatment has limited the overall effectiveness of HCT. In addition, disease relapse also limits overall success. Novel strategies are needed which can suppress the development of GVHD but still maintain an effective immune response to provide a GVT effect. It has been shown that NK cells have the capability of suppressing the development of GVHD while inducing an anti-tumor response. The trafficking of cells to specific organs and tissue sites after transplant can be a major factor in determining whether or not they contribute to GVT and GVHD reactions. Little is known about the trafficking patterns of NK cells following hematopoietic cell transplantation, their proliferation or how long they persist in vivo. We investigated the trafficking patterns of NK cells in vivo in an allogeneic and syngeneic bone marrow transplant setting using a novel in vivo bioluminescence imaging (BLI) technique. Freshly isolated NK cells from FVB L2G85 transgenic mice, which constitutively express the luciferase gene and can be imaged by BLI, were transplanted along with T-cell depleted bone marrow into lethally irradiated BALB/c (allogeneic) or FVB (syngeneic) mice. BLI of the irradiated mice on successive days post-transplant indicated that in the allogeneic setting, NK cells traffic to distinct lymphoid organs, such as the cervical lymph nodes and spleen. The bioluminescent signal was 2.75-fold greater on Day 6 post-transplant than on Day 2 post-transplant, indicating a significant in vivo expansion of the NK cells in an allogeneic recipient in the first week post-transplant. In the syngeneic setting, NK cell trafficking to distinct lymphoid organs was not observed, and the bioluminescent signal intensity emitted from the transplanted mice remained constant. Thus, we anticipate that the in vivo expansion of NK cells seen in the allogeneic bone marrow transplant setting is not due to homeostatic proliferation alone. Continuing studies will address whether the in vivo expansion is driven by NK cell receptor-ligand interactions or MHC Class I differences. Understanding NK cell trafficking and proliferation could provide novel insights into enhancing function of both innate and adoptively transferred NK cells.
Vaccination of Advanced Prostate Cancer Patients with PSCA and PSA Peptide-Loaded Dendritic Cells Induces Cellular Immune Responses That Correlate with Superior Overall Survival.
Authors: Kaskel Anna K. , Zeiser Robert , Jochim Rosa , Schultze-Seemann Wolfgang , Waller Cornelius , Veelken Hendrik
Published: April 2020

Prostate stem cell antigen (PSCA) and prostate-specific antigen (PSA) are overexpressed in most prostate cancers. PSCA- and PSA-derived, HLA-A2 binding peptides are specific targets for T cell responses in vitro. A phase I/II trial was performed to demonstrate feasibility, safety, and induction of antigen-specific immunity by vaccination with dendritic cells (DC) presenting PSCA and PSA peptides in patients with hormone- and chemotherapy-refractory prostate cancer. Patients received four vaccinations with median of 2.7x10e7 peptide-loaded mature DC s.c. in biweekly intervals. Twelve patients completed vaccination without relevant toxicities. Six patients had stable disease (SD) after four vaccinations. One patient had a complete disappearance of lymphadenopathy by ultrasound despite rising PSA. Regarding PSA kinetics, in one patient, the log PSA slope became negative after treatment, and three patients had significant decreases in the log PSA slopes. However, duration of response was short, with a median time to progression of 65 days (12–223). Four patients with SD and one progressor developed a positive DTH after the 4th vaccination. With a median survival of all patients of 13.4 months, DTH-positivity was associated with significantly superior survival (22 vs. 8 months, p=0.003). HLA tetramer analysis detected high frequencies of peptide-specific T cells after two vaccinations in one patient who was also the sole responder to concomitant hepatitis B vaccination as an indicator of immune competence and survived 27 months after start of vaccination. Taken together, vaccination with PSA/PSCA peptide-loaded, autologous DCs may induce cellular responses primarily in immunocompetent patients which were associated with superior outcome. The retardation of PSA kinetics upon vaccination suggest a causal influence of the treatment on the natural course of the disease. Testing of DC-based vaccination is warranted for patients at earlier stages of prostate cancer.
Effect of Treatment with Rituximab in Patients with Refractory Autoimmune Cytopenias.
Authors: Dungarwalla Moez , Marsh Judith , Tooze Jenny , Lucas Geoff , Ouwehand Willem , Pettengell Ruth , Dearden Clare E. , Gordon-Smith Edward C. , Elebute Modupe O.
Published: April 2020

The autoimmune cytopenias encompass the disorders of immune thrombocytopenia purpura (ITP), autoimmune haemolytic anaemia (AIHA), autoimmune neutropenia (AIN), pure red cell aplasia (PRCA) and various combinations of these conditions. We describe 34 patients with severe autoimmune cytopenias resistant to standard immunosuppression treated with the anti-CD20 monoclonal antibody Rituximab. 17 patients had ITP, 6 AIN, 2 AIHA, 2 PRCA, 4 AIHA and ITP (Evan’s syndrome), 1 AIHA and AIN, 1 PRCA and ITP, and 1 AIN and ITP. 28 patients had primary autoimmune cytopenias and in 6 patients, the cytopenia was secondary to an underlying disorder (3 B-NHL, 1 T-NHL, 1 B-CLL and 1 SLE).

Rituximab was administered intravenously at a dose of 375mg/m2 weekly for 4 weeks. 33 of 34 patients completed treatment. In one patient treatment was discontinued after 3 infusions due to a hypotensive syncopal episode. No other major infusion related side effects were observed. Responses were seen in 9/17 patients with ITP, 2/2 patients with AIHA and 4/4 patients with AIHA and ITP. Responses were sustained in 8/9 ITP patients with median follow up of 25.8months (range 4–66 months). We observed a complete response in 5 of 7 ITP patients who were previously splenectomised.

In contrast, there was no response in 5/6 patients with AIN and in 3/3 patients with PRCA.

In two patients we observed severe neutropenia at 3 months post Rituximab which was associated with fatal infection in both patients, namely pnemocystis pneumoniae infection and acute exacerbation of bronchiectasis. Apart from these 2 cases no other infections were seen.

In conclusion, Rituximab appears to be a an alternative and safe option in the treatment of patients with ITP, AIHA and Evan’s syndrome inducing sustained clinical remissions in patients who have failed to respond to conventional immunosuppression and/or splenectomy. In contrast Rituximab does not seem to be effective in AIN and PRCA and this may reflect a different pathogenesis for these conditions.
Gene Expression Analysis Uncovers Possible Resistance Mechanisms to Bortezomib in Mantle Cell Lymphoma That Differ from Mechanisms Identified in Other Lymphoid Malignancies.
Authors: Rizzatti Edgar G. , Mora-Jensen Helena I. , Billings Eric , Lai Raymond , Wiestner Adrian
Published: April 2020

Bortezomib, a potent inhibitor of the 26S proteasome, has remarkable activity against some lymphoid malignancies, particularly multiple myeloma and mantle cell lymphoma (MCL). The observed 30–50% clinical response rate to bortezomib as a single-agent in relapsed MCL is believed to be mediated mainly by inhibition of the NFkB signaling pathway. Recently, Hsp27 has been shown to confer resistance to bortezomib in a lymphoid cell line. However, much less is known about the mechanisms underlying anti-tumor activity of or resistance to bortezomib in MCL. To address these questions we studied 10 MCL cell lines with t(11;14)(q13;q32) as in vitro models. IC50 values were measured by the MTT cytotoxicity assay for both bortezomib and the BAY 11-7082 compound, a specific inhibitor of the NFkB pathway. The cell lines showed different profiles of response to bortezomib and were grouped according to their IC50 values as sensitive (S) (Granta 519, Jeko-1, SP-49, UPN-1), intermediate (In) (HBL-2, JVM-2, Z-138) and resistant (R) (Mino, NCEB-1, SP-53). The mean IC50 for bortezomib in the R group was 3 times higher than the mean IC50 of the S group. The cell lines also showed very different profiles of response to the NFkB-specific inhibitor BAY 11-7082, but grouped differently, suggesting that other mechanisms, in addition to the NFkB pathway, participate in the anti-neoplastic effect of proteasome inhibition in MCL. Resistance to both drugs had no correlation with P-glycoprotein activity as measured by the rodhamine efflux assay. We performed gene expression profiling on Affymetrix U133A 2.0 arrays of all 10 cell lines. Using GeneSpring software (Agilent), we normalized expression to the mean of the S group and identified 79 transcripts that showed a dose response behavior; that is, genes whose mean expression was 1.5x higher in the In versus the S group and 1.5x higher in the R versus the In group. Conversely, 55 transcripts followed the opposite trend, being down-regulated at least 1.5x in both comparisons. This list contained genes important in apoptosis control and in transmembrane transport, but was most notable for an overexpression of stress response genes in the R group. Notably, expression of Hsp70 was almost 10-fold higher in the R versus S group. We therefore analyzed expression of the HSP70 as well as HSP27 and HSP90 proteins by Western blotting. Hsp70 protein was highly expressed in all MCL cell lines and there was no discernable correlation with bortezomib resistance for any of the HSP-proteins analyzed. Other resistance mechanisms appear more important in MCL and we are currently testing further candidate genes for their ability to confer resistance to bortezomib in MCL.
Co-Treatment with the hsp90 Inhibitor 17-Dimethylaminoethylamino-17-Demethoxygeldanamycin (DMAG) and Histone Deacetylase Inhibitor (HDI) Vorinostat (Suberoylanilide Hydroxamic Acid, SAHA): A Highly Active Combination Against Human Mantle Cell Lymphoma (MCL) Cells.
Authors: Hatter Angela , Bali Purva , Balasis Maria , Fiskus Warren , Boyapalle Sandhya , Kumaraswamy Sandhya , Rocha Kathy , Pranpat Michael , Hannah Alison , Sotomayor Eduardo , Richon Victoria , Bhalla Kapil
Published: April 2020

We have previously reported that agents that inhibit ATP binding and chaperone function of hsp90 are highly active against wild type and mutant Bcr-Abl and mutant FLT-3 containing human acute leukemia cells. In the present studies, we determined the effects of a more soluble and potent geldanamycin analogue, DMAG (Kosan Biosciences Inc.), and/or hydroxamate histone deacetylase inhibitor (HHDI), vorinostat (Merck & Co., Inc.), against human MCL Jeko1 and MO2058 cells. These cells contain the characteristic MCL-associated chromosomal translocation t(11; 14)(q13;q32), which results in the overexpression of cyclin D1. Recently, HHDIs, such as vorinostat, have been shown to inhibit HDAC6, which results in the acetylation of hsp90 and inhibition of its ATP binding and chaperone function. Treatment with vorinostat (0.5 to 2.0 μM) induced the accumulation of the cells in the G1 and DMAG (0.1 to 0.5 μM) in the G2/M phase of the cell cycle. Both agents induced apoptosis in a dose-dependent manner (up to 50%). While vorinostat induced both p21 and p27 levels, DMAG only increased the intracellular levels of p21. Treatment with either agent depleted the intracellular levels of c-Myc, c-Raf, Akt and cdk4 in a dose dependent manner. It is well established that the chaperone association with hsp90 maintains Akt, c-Raf, cyclin D1 and cdk4 in the native and active conformation, and inhibition of hsp90 promotes their polyubiquitylation and proteasomal degradation. Notably, co-treatment with DMAG (e.g., 0.25 μM) and vorinostat (e.g., 2.0 μM), more than either agent alone, markedly attenuated the levels of cyclin D1 and cdk4, as well as the levels of c-Myc, c-Raf and Akt. The combination of DMAG and vorinostat also induced significantly more apoptosis of Jeko1 and MO2058 cells, as compared to the treatment with either agent alone (p < 0.01). These findings demonstrate that the combined treatment with vorinostat and DMAG is highly active against human MCL cells, and support the rationale to determine the in vivo efficacy and safety of the combination against human MCL.
Chloroquine Inhibits Autophagy, Enhances p53-Dependent Apoptosis, and Delays Tumor Recurrence in a Mouse Model of B Cell Lymphoma.
Authors: Amaravadi Ravi K. , Yu Duonan , Thomas-Tikhonenko Andrei , Thompson Craig B.
Published: April 2020

Burkitt’s lymphoma is an example of an aggressive B cell neoplasm characterized by overexpression of the c-myc oncogene and frequent inactivation of the tumor suppressor gene p53. A non-transgenic mouse model of Burkitt’s lymphoma was generated by retroviral transduction of the human c-myc gene into bone marrow cells derived from the p53-estrogen receptor (p53ER) knock-in mouse. The resulting myc/p53ER cells produce an aggressive B cell lymphoma when injected subcutaneously into the flanks of syngeneic mice. When tumor-bearing mice are treated with tamoxifen intraperitoneally, the p53ER fusion protein is targeted to the nucleus where p53-dependent apoptosis can take place. On successive in vivo passages, cells develop the ability to survive p53 activation and escape p53ER-dependent apoptosis despite tamoxifen treatment and nuclear localization of the p53ER fusion protein. We hypothesized that cells resistant to p53-dependent apoptosis utilize autophagy as an essential survival mechanism. Thus, these tumors could be sensitive to chloroquine, a lysosomotropic inhibitor of autophagy that has been used extensively in humans as an antimalarial and for the treatment of rheumatoid arthritis. Daily intraperitoneal chloroquine or hydroxychloroquine treatment of mice bearing myc/p53ER tumors in the absence of tamoxifen resulted in a delay in tumor growth. When tamoxifen was added to induce nuclear localization of p53ER, mice that received tamoxifen plus chloroquine had a complete tumor response while mice that received tamoxifen plus saline had transient tumor shrinkage followed quickly by regrowth. Tamoxifen plus chloroquine treatment enhanced the expression of p53-dependent target genes and increased caspase activation compared to tamoxifen plus saline treatment. A higher percentage of cells in tumors treated with tamoxifen plus chloroquine underwent apoptosis compared to tumors treated with tamoxifen plus saline. Moreover, tumors that recurred in the mice treated with daily tamoxifen plus chloroquine did so after a significantly longer latency period then mice treated with tamoxifen plus saline. Recurrent tumors showed loss of expression of p53 target genes. Electron microscopy of recurrent tumors confirmed the accumulation of vacuoles in chloroquine treated tumors compared to controls, suggesting inhibition of lysosome function leads to the accumulation of ineffective autophagic vacuoles. These results indicate that inhibiting autophagy with lysosomotropic chloroquine derivatives could be a useful therapeutic addition to treatment regimens for aggressive B cell lymphomas.
Polymorphisms in FcγRIIIA Are Genetically Linked to FcγRIIA and May Account for the Primary Predictive Role Ascribed to Polymorphisms in FcγRIIIA-158 in Determining Rituximab Responses.
Authors: Hatjiharissi Evdoxia , Hansen Mark , Verselis Sigitas , Santos Daniel Ditzel , Xu Lian , Manning Robert , Hunter Zachary , Leleu Xavier , Ho Allen W. , Patterson Christopher J. , Fox Edward , Treon Steven P.
Published: April 2020

γWe and others have previously described polymorphisms in codon 158 and 48 for FcγRIIIα as predictors of clinical responses to the anti-CD20 monoclonal antibody rituximab in patients with low-grade non-Hodgkin’s lymphoma, including Waldenström’s Macroglobulinemia. The expression of at least one valine (V) at FcγRIIIα-158 and Leucine/Histidine (L/H) at FcγRIIIα-48 is associated with higher response rates to rituximab, though primacy for FcγRIIIα-158 over 48 polymorphisms for determining rituximab response is attributed to genetic linkage. As part of these studies, we sought to determine if polymorphisms in FCγRIIA were linked to FCγRIIIA since higher response rates have been observed in patients who express Histidine/Histidine (H/H) in FcγRIIA-131. We therefore performed sequence analysis of the whole FcγRIIa gene and exons 3 and 4 of the FcγRIIIa gene in 52 healthy donors. The genetic association between polymorphisms was calculated using the statistical program GOLD and expressed as linkage disequilibrium (D’). Single nucleotide polymorphisms were commonly found in FcγRIIIa at codons 158, 48 and in FcγRIIa at codons 131, 27. The genotype frequencies for polymorphisms are as follows: FcγRIIIa-158: 25% F/F; 63% F/V; 12% V/V; FcγRIIIa-48: 67% L/L; 10% L/R; 17% L/H; FcγRIIa-131: 29% H/H; 46% H/R; 25% R/R; FcγRIIa-27: 71% Q/Q; 27% Q/W; 2% W/W. Strong linkage was found between the FcγRIIIa-158 and −48 (D’=1, p=0.001), as well as between FcγRIIa-131 and -27 (D’=1, p=0.00082). Specifically, the expression of F at FcγRIIIa-158 was found to be associated with expression of L at FcγRIIIa-48, whereas the expression of H at FcγRIIa-131 was associated with the expression of Q at FcγRIIa-27. Importantly, we observed links among polymorphisms at FcγRIIa and FcγRIIIa. Specifically, FcγRIIIa-48 was linked to FcγRIIa-27 (D’=0.765, p<0.00001) and FcγRIIa-131 (D’=1, p=0.0049), since all donors who were FcγRIIIa-48 L/L donors were also FcγRIIa-27Q/Q and -131R/R, respectively. Lastly, we also observed linkage between FcγRIIIa-158 and FcγRIIa-27 (D’=1, p=0.0008). Our results demonstrate significant genetic association between polymorphisms in FcγRIIa and FcγRIIIa genes, and may account for the primary predictive role ascribed to polymorphisms in FcγRIIIa-158 in determining rituximab responses.
Rituximab Maintenenance Therapy in CD20+ B-Cell Non-Hodgkin-Lymphoma - First Interim Results of a Prospective Randomised Phase II Study.
Authors: Witzens-Harig Mathias , Hensel Manfred , Neben Kai , Benner Axel , Schmier Johann W. , Dreger Peter , Kraemer Alwin , Ho Anthony D.
Published: April 2020

Clinical and pharmacokinetic data suggest that the effect of rituximab could be improved by prolonged exposure to the drug. To test for this hypothesis we performed a prospective randomized trial of rituximab maintenance therapy in patients with CD20+ B-cell Non-Hodgkins-Lymphoma. After completion of standard treatment patients were randomized to either observation or maintenance therapy with rituximab (375 mg/m2) every 3 months for 2 years. Patients with aggressive lymphoma were enrolled if they had achieved a complete response (CR) after initial treatment. Patients with aggressive lymphoma with residual tumor mass were examined with positrone emission tomography (PET) and qualified for randomization if PET showed no signs of tumor activity. Patients with indolent lymphoma qualified for the study if at least a partial response (PR) was achieved. So far 87 patients (pts) with CD20+ B-cell Non-Hodgkins-Lymphoma were enrolled in this trial. Histological subtypes included diffuse large cell lymphoma (38 pts), follicular lymphoma (17 pts), mantle cell lymphoma (16 pts), primary mediastinal lymphoma (11 pts), marginal zone lymphoma (1 pt), Burkitt’s lymphoma (2 pt), primary intestinal lymphoma (1pt) and unclassified B-cell lymphoma (1 pt). No severe adverse events were observed during rituximab maintenance therapy. We conclude that rituximab maintenance therapy is feasable, safe and well tolerated in patients with CD20+ B-cell Non-Hodgkins-Lymphoma. First results from an interim analysis including event free survival and overall survival for the observation group and for the maintenance therapy group will be presented.
Rituximab Retreatment in B-Cell Non-Hodgkins Lymphoma Patients.
Authors: Bairey Osnat , Dann Eldad J. , Herishanu Yair , Ruchlemer4 Rosa , Tadmor5 Tamar , Gavish6 Israel , Volchek7 Yulia , Yuklea8 Mona , Bulvik9 Shlomo , Ben-Shahar10 Menachem , Levy11 Itai , Rosenbaum Hanna , Quitt12 Miriam , Solomon13 Liora , Shpilberg Ofer ,   Israel Rituximab Retreatment Study Group
Published: April 2020

Rituximab has revolutionized the treatment outcome of non-Hodgkins lymphoma (NHL) patients but its role in retreatment in all NHL patients is not yet established. Therefore, the Israel Rituximab Retreatment Study Group was organized in order to summarize retrospectively the treatment results of B-cell NHL patients who received ≥ 2 rituximab treatments. Eighty-eight NHL patients from 13 medical centers were enrolled, 39 males, 49 females with a mean age of 57.3 years (range 26–88). Sixty-four patients (73%) had indolent lymphoma (21- follicular grade I, 17 follicular grade II, 10-follicular grade III, 9-small lymphocytic, 6- MALT, 2- marginal zone, 1-lymphoplasmacytic). Eighteen patients (20%) had aggressive lymphoma [diffuse large B-cell lymphoma (DLBCL)] and 6 patients had mantle cell lymphoma. Fifty-nine patients received one regimen of chemotherapy and 23 patients received 2 chemotherapy regimens before the first treatment with rituximab. There was no significant difference in the median time to progression (TTP) after the first rituximab treatment whether the patient received no previous chemotherapy or 1 or 2 previous chemotherapy regimens (12 vs 12 vs 14 months, respectively). All patients received 2 courses of rituximab, 33 patients-3 courses, 6 patients -4 courses and 2 patients-5 courses, with a mean of 4.4 doses in each course (range 1–8). Only 45 patients received any treatment after the second course of rituximab. The first course of rituximab was administered alone in 46 patients and with chemotherapy in 42 (24 CHOP, 6 COP, 6 FC, 2 chlorambucil, 1 LMP, 1 DVIP, 1 ESHAP, 1 MACOP-B). There was no difference in the median time to next treatment (TTNT) whether the rituximab was given alone or with chemotherapy (16 and 14 months, respectively). The second course of rituximab was administered alone in 46 patients and with chemotherapy in 42 patients (13 CHOP, 11 FC, 6 COP, 5 ICE, 3 ESHAP, 2 HyperCVAD, 1 CNOP 1 VEEP). The addition of chemotherapy did not change the overall response (CR and PR) to the second rituximab treatment (72% in rituximab alone vs. 69% in rituximab with chemotherapy, in indolent lymphoma patients 76% vs. 72%, in aggressive lymphoma patients 70% vs. 62.5%, respectively). TTP after the second rituximab treatment was similar or possibly longer than after the first treatment (median 14 and 12 months, mean 23.4 and 16.2 months respectively).

Conclusions: The response to a second rituximab treatment is the same whether the rituximab is given alone or combined with chemotherapy. TTP after retreatment with rituximab is as good as after the first treatment. Prospective studies should examine the treatment benefit of adding chemotherapy to a second treatment with rituximab.
Re-Treatment with Rituximab Plus Chemotherapy in Patients with Diffuse Large-B Cell Lymphoma (DLBCL): A Spanish Multicenter Study.
Authors: Canales Miguel A. , Palacios A. , Martinez-Chamorro C. , Cruz M. A. , Moraleda J. M. , Terol M. J. , Almagro M. , Garcia-Vela J. A. , Aguiar D. , Caballero D. , Sancho J. M. , Ferrer S. , Rueda A. , Calvo J. M. , Fernandez J. A. , Diaz-Mediavilla J. , Gonzalez J. , Mayans J. , Montesinos C. , Rios E. , Ruedas A. , Sabin P. , Fernandez-Jurado A.
Published: April 2020

Rituximab in combination with CHOP-like chemotherapy has demonstrated to improve the results in young and elderly patients with DLBCL compared to chemotherapy alone. Re-treatment with rituximab is feasible in patients with indolent lymphoma, but there is few data on the effect of rituximab in combination with chemotherapy in the re-treatment of relapsed patients with DLBCL who have respond previously to rituximab + chemotherapy. In order to investigate the effect of rituximab in this setting, we have performed a multicenter retrospective study in 50 patients with DLBCL re-treated with rituximab in combination with chemotherapy. We have included in this study 46 patients (31 males, 15 females), median age 60 (range, 20 to 81 years), who achieved CR with the previous rituximab-containing regimen. At initial diagnosis, 32 patients had Ann-Arbor stage III or IV and 20 patients had IPI 3 or 4. The median interval between courses was 14.7 months. The most frequent regimens administered as up-front therapy in combination with rituximab were CHOP-like regimens (30 patients) and the commonest chemotherapy regimens used as salvage therapy were R-ESHAP (20 patients), R-ICE (7 patients), rituximab in combination with gemcitabine-based regimens (4 patients) and TTR (3 patients). The overall response rate in the assessable population was 81% (46% CR, 35% PR). In 25 assessable patients who have received rituximab in combination with chemotherapy as second-line therapy, the overall response rate was 92% (56% CR, 36% PR). The overall response rate was slightly lower in those patients re-treated with rituximab as third-line therapy (78% with 33% CR and 45% PR). The follow-up is too shorter (median 6 months) to assess the impact of re-treatment on duration of remission. The adverse events were not different to those usually observed with these regimens. In conclusion, the re-treatment with rituximab in relapsed patients with DLBCL who had respond previously to rituximab in combination with chemotherapy seems not to compromise the results in terms of response. Logically, larger prospective studies are necessary to confirm these encouraging results.
CDDO Increases Translation of CCAAT Enhancer Binding Protein alpha To Induce Granulocytic Differentiation.
Authors: Koschmieder Steffen , D′Alo′ Francesco , Radomska Hanna , Kobayashi Susumu , Levantini Elena , Suh Nanjoo , Sporn Michael B. , Di Ruscio Annalisa , Voso Maria Teresa , Berdel Wolfgang E. , Müller-Tidow Carsten , Serve Hubert , Tenen Daniel G.
Published: April 2020

The triterpenoid 2-Cyano-3,12-dioxooleana-1,9-dien-28-oic acid (CDDO) is a novel antineoplastic drug which induces apoptosis of a wide variety of tumor cells in vitro and in vivo and leads to granulocytic differentiation of hematopoietic progenitor cells. We studied the effect of CDDO on CCAAT enhancer binding protein alpha (CEBPA), a transcription factor which is critical for granulocytic differentiation. In HL60 myeloblastic cells, CDDO (0.01 to 2 uM) dose-dependently decreased the number of cells in culture and increased the fraction of apoptotic cells. However, at doses which did not induce apoptosis, CDDO increased the number of granulocytic cells, as assessed by morphology, NBT assay, and FACS, and Northern blotting showed an increase of GCSFR and a decrease of c-myc mRNA. Phagocytosis of FITC-labeled E. coli bacteria by these cells was enhanced by CDDO. While CEBPA mRNA was decreased, CEBPA protein was significantly increased within 24 hours of treatment, and this was not abrogated by preincubation with the caspase inhibitor Z-DEVD-fmk, again suggesting that these effects were independent of apoptosis. CDDO increased the ratio of the transcriptionally active isoform p42 and the inactive p30 isoform 3-fold, and gel shift assays showed enhanced DNA binding to a GCSFR promoter probe. Since eukaryotic translation initiation factors (eIF) have been described to alter the CEBPA protein isoform ratio, we studied the effects of CDDO on eiF2 alpha and eiF4E activity. CDDO increased the phosphorylation of eIF4E and decreased the phosphorylation of eIF2 alpha within 5 hours of treatment, and this was associated with an increase of the p42/p30 CEBPA ratio. In the presence of the translation inhibitor cycloheximide, CEBPA protein levels decreased after 2 hours, suggesting that CDDO did not stabilize CEBPA and that de novo protein synthesis was required for the observed effects. The effect of CDDO on the p42/p30 ratio was mimicked by 2-AP, which inhibits eIF2 alpha phosphorylation, but was independent of PPARgamma and TGFß pathways, as demonstrated by preincubation with GW9662, or TGFß1, respectively. In primary blasts from patients with acute myeloid leukemia (AML), the p42/p30 ratio of CEBPA was enhanced by CDDO treatment. In conclusion, CDDO leads to granulocytic differentiation and translational induction of CEBPA protein. Since CEBPA function is impaired in many patients with AML, CDDO may provide a novel treatment approach for these patients.
Stroma-Activated Integrin-Linked Kinase (ILK) Supports Survival of Leukemic Cells Via Stimulation of Notch-Hes Signaling: New Therapeutic Opportunities.
Authors: Tabe Yoko , Jin Linhua , Tanaka Nobuko , Andreeff Michael , Konopleva Marina
Published: April 2020

We have previously demonstrated that the BM microenvironment plays a crucial role in the pathogenesis of AML by influencing tumor growth, survival, and drug resistance. Integrin-linked kinase (ILK) has been shown to directly interact with β integrins and phosphorylate AKT in a PI3-kinase (PI3K)-dependent manner to promote cell survival and proliferation. HES-1 encodes a basic helix-loop-helix transcription factor downstream of the Notch receptor, and functions as a positive regulator of hematopoietic and neuronal stem cell self-renewal. Direct co-culture of human mesenchymal stem cell (MSC) and leukemic NB4 cells results in activation of PI3K/ILK/AKT signaling (elevated phospho(p)-Akt, p-GSK3β and nuclear-localized β-catenin), increased expression of Notch1 and Hes1 proteins and upregulation of p-STAT3 detected by Western blot and confocal microscopic analyses. Both, PI3K inhibitor LY294002 (20 μM) and ILK inhibitor QLT0254 (10 μM) specifically inhibited stroma-induced activation of AKT and Stat-3 signaling, suppressed GSK phosphorylation and decreased Notch 1 and HES1 expression. This resulted in massive induction of apoptosis which was not abrogated by stromal co-culture (AnnexinV positivity %, MSC(-) vs MSC(+); control 33.8±2.5 vs 27.3±1.9 p=0.02, QLT 51.4±2.5 vs 55.8±3.5 p=0.26, LY 47.0±8.1 vs 47.9±6.1 p=0.85, 48hrs). In contrast, GSK3b inhibitor BIO (0.1 μM) prevented the serum-withdrawal-induced apoptosis of NB4 cells (AnnexinV positivity %, control 38.1±4.0 vs BIO 25.9±3.4 p=0.003, 48hrs) with marked increase in Notch1 and Hes1 expression detected by confocal microscopy. These observations indicate that Notch signaling is involved in leukemic cell survival stimulated by BM stromal interactions via activation of the ILK-AKT-GSK3β pathway. We have next investigated the effects of leukemic cells on stroma cells. Coculture with NB4 cells caused significant increase in Hes1 and Bcl2 proteins in MSC along with phosphorylation of STAT3 and Akt, which were all abrogated by the treatment with QLT0254 or LY294002. In summary, these results demonstrate that interactions of leukemic and bone marrow stromal cells result in activation of PI3K/ILK/AKT and Notch-Hes signaling in both, leukemic and stromal cells. Disruption of these interactions by specific ILK inhibitors represents a novel therapeutic approach to eradicate leukemia in the bone marrow microenvironment via direct effects on leukemic cells and by targeting activated bone marrow stromal cells.