bluuebunny/crossref_metadata_2025 huggingface dataset:
HIV-1 Proviral Integration Is Blocked by Agonists to the VpPAC2 Neuroendocrine Receptor.
Authors: Branch Donald R. , Bokaei Payman Baradar , Sakac Darinka , Ma Xue-Zhong
Published: April 2020

VPAC1 is a 7-transmembrane G-protein-coupled neuroendocrine receptor previously shown to transduce a facilitation signal for HIV-1 infection (Branch DR, et al., AIDS.2002;16:309–319). VPAC2, a related receptor, has been reported to have opposing function when compared to VPAC1 (Xia M, et al., J Immunol.1996;157:1132–1138; Tsutsumi M, et al., Diabetes.2002;51:1453–1460). We therefore examined whether stimulation of VPAC2, in contrast to VPAC1, may act to inhibit HIV-1 infection. Using three different and specific agonists of VPAC2, helodermin, RO 25-1553, and R3P55, daily treatment with low concentrations (10−9M) resulted in ~75% to 95% inhibition of either X4 or R5 HIV-1 productive infection in cell lines or primary peripheral blood mononuclear cells. The agonists VIP, PACAP, and secretin, that stimulate the two other VPAC receptor family members VPAC1 and PAC1, did not inhibit HIV-1 infection. Also, Hut78 cells, that lack VPAC2 and are infected by HIV-1, show no effect of VPAC2 agonists on HIV-1 infection. However, Hut78 cells transfected with human VPAC2 cDNA to overexpress this receptor become resistant to HIV-1 infection when treated with VPAC2 agonists.

VPAC2-mediated inhibition of productive HIV-1 infection was not due to effects on CD4 or chemokine co-receptor expression, cell growth, or apoptosis. Treatment with VPAC2 agonists also did not inhibit HIV-1 entry into the host cell. However, compared to untreated or cells treated with VPAC1-specific agonists, VPAC2 stimulation profoundly inhibits HIV-1 proviral DNA integration into the host genome. The block in integration was found to be due to the ability of agonists to VPAC2 to inhibit formation of 2-LTR circles, required for HIV-1 integration within the nucleus. We conclude that VPAC2-specific agonists are strongly inhibitory for productive HIV-1 infection. Furthermore, this inhibition is mediated by suppression of 2-LTR circle formation preventing proviral integration. Agonists of VPAC2 appear to be excellent candidates for future development as possible drugs for the amelioration of treatments aimed at the prevention of HIV/AIDS.
Selective Inhibition of Receptor Pathways for Macrophage-Specific Cytokines by HIV-1 Nef Protein.
Authors: Okada Seiji , Harada Hideki , Suzu Shinya
Published: April 2020

HIV-1 Nef protein is a well-known major determinant of the pathogenicity of the virus and much attention has been given to Nef to explain its contribution to HIV-1 pathogenesis. One of the important features of Nef is the binding to a variety of host intracellular proteins. Hck, a member of Src family tyrosine kinases, is one of the binding partners of Nef. Meanwhile, Src kinases including Hck are key regulators for signaling pathways of several cytokine receptor systems. We therefore investigated whether Nef would affect cytokine receptor signaling pathways. Both GM-CSF and M-CSF are primary cytokines for monocytes/macrophages, and regulates their development and effector functions. We introduced a conditionally active Nef (Nef-estrogen receptor hormone binding-domain fusion protein) into the human leukemia cell line of which proliferation was dependent on those cytokines, and analyzed their ligand-dependent proliferation and receptor signaling events. Nef activation did not apparently affect the proliferation mediated by GM-CSF but markedly caused the inhibition of proliferation mediated by M-CSF, indicating that Nef selectively inhibited the M-CSF receptor pathways. In fact, Nef inhibited the tyrosine-phosphorylation/activation of M-CSF receptor and the molecular association with the activated receptor, which was the earliest signaling events induced by M-CSF. We found that Nef-activated Hck constitutively associated with M-CSF receptor complex. Since the formation of the molecular complex should occur under physiological conditions, i.e., upon M-CSF stimulation, the constitutive association occurred in Nef-active cells was an aberrant interaction. These results suggested that HIV-1 Nef interfered with M-CSF receptor signaling through Hck activation and thereby inhibited M-CSF bioactivity. The experiments in which Nef, Hck and M-CSF receptor were transiently expressed within 293 T cells in various combinations supported the notion (Suzu S et al., Blood 2005). The selective inhibition of Nef on cytokine signaling might result in the modification of macrophage functions such as cytokine/chemokine production, creating an immunologic environment favorable for HIV-1 virus production.
Hodgkin’s Disease and HIV Infection (HD-HIV) in Pre- and Post-HAART Era: The GICAT (Italian Cooperative Group on AIDS and Tumors) Experience in 139 Patients.
Authors: Tirelli Umberto , Vaccher Emanuela , Rossi Giuseppe , Schiantarelli Clara , Fasan Marco , Spina Michele
Published: April 2020

Background: Hodgkin’s disease (HD) in HIV infected patients shows aggressive tumour behaviour including a high frequency of unfavourable histologic subtypes, advanced stage and extranodal involvement and a poor outcome. The introduction of highly active antiretroviral therapy (HAART) has opened a new prospective in the treatment of patients with HD-HIV.

Methods: We analysed the results of four consecutive prospective phase II studies carried out within the GICAT since 1988.

Results: Table 1 summarizes the results of the prospective studies.

Conclusions: Since the widespread use of HAART, CR rate is improving and OS and DFS as well. The concomitant use of HAART and chemotherapy is feasible and advisable in our experience. In the HAART era HD seems to be a more successfully treatable malignancy, although at higher incidence and probably with the same aggressive behaviour at presentation as in the pre-HAART era.

Table 1: Results REGIMEN # of PTS HAART + CT STAGE III–IV CR RATE OS DFS EBV 17 No 88% 53% 11 mos -- EBVP 35 No 83% 74% 16 mos 53% at 3-yrs STANFORD V 59 Yes 71% 81% 68% at 2-yrs 70% at 2-yrs VEBEP 28 Yes 68% 75% 86% at 2-yrs 90% at 2-yrs
Gain of Chromosome 21 Is Associated with Early Treatment Sensitivity in Childhood Acute Lymphoblastic Leukemia.
Authors: Steinemann Doris , Cario Gunnar , Stanulla Martin , Tauscher Marcel , Hinrichsen Tanja , Michaelsen Gudrun , Viehmann Susanne , Lichter Peter , Schrappe Martin , Schlegelberger Brigitte
Published: April 2020

Acute lymphoblastic leukemia (ALL) is the most common malignancy in childhood. In vivo response to initial therapy, as assessed by determination of minimal residual disease (MRD) at 5 and 12 weeks of treatment, has evolved as the strongest prognostic factor in pediatric ALL patients treated according to the BFM regime. In this study, ten patients with a poor treatment response (MRD load ≥ 10−3 at week 12, MRD-high risk, HR) and five treatment-sensitive (no measurable MRD at weeks 5 and 12, MRD-standard risk, SR) patients were investigated by means of high-resolution bacterial artificial chromosome (BAC) array-based comparative genomic hybridization (array-CGH). To ensure homogeneity with regard to prognostic factors, the following inclusion criteria were used: B cell precursor or common ALL, DNA index of 1.0, no BCR/ABL, no MLL/AF4, no TEL/AML1 rearrangements. A gain for all chromosome 21-related BAC clones was observed in all SR patients. None of the ten HR patients showed a gain of any region of chromosome 21. This result could be confirmed by means of fluorescence in situ hybridization using the LSI AML1/ETO DC probe set. Besides the basic level of chromosome 21 gain, higher levels of gain or amplification observed for individual regions were found in three SR patients. Recurrent genomic alterations in the group of HR patients were loss of chromosomal region 2p11.22 (9/10), a gain of 8q24.13 (7/10) and loss of 14q32 (8/10). To see whether these findings may help to further subdivide the intermediate risk group patients (any MRD positivity at week 5, MRD load < 10−3 at week 12) we additionally analyzed three patients of this group: two patients in long-term complete remission and one patient who relapsed 29 months after diagnosis. Again, a gain for all chromosome 21-related clones was detected in both patients in complete remission. In contrast, the leukemic sample of the relapsed patient demonstrated no gain of chromosome 21. We conclude that array CGH analysis in childhood ALL may lead to the identification of additional prognostic markers. The relevance of the observed gain of chromosome 21 has to be validated in a larger set of patients.
A Plateau after 30 Months of Follow-Up in B -Lineage Childhood Acute Lymphoblastic Leukemia (ALL) with t(1;19)(q23;p13)/E2A-PBX1?.
Authors: Baruchel André , Chevret Sylvie , Auvrignon Anne , Ballerini Paola , Michel Gérard , Gabert Jean , Vermylen Christiane , Perel Yves , Bilhou-Nabera Chrystèle , Lejars Odile , Schmitt Claudine , Jonveaux Pierre , Soulier Jean , Vandenaker Jacqueline , Auclerc Marie-Françoise , Leblanc Thierry , Leverger Guy ,   for the FRALLE Group
Published: April 2020

Late events are observed in B-lineage ALL with no plateau seen if a sufficient observation period is provided. The prognosis of childhood ALL associated to the t(1;19)(q23;p13)/E2A-PBX1 translocation has improved over the two last decades. The purpose of this study is to define the kinetics of relapse in a large cohort of children and to raise the possibility of a plateau in that entity. We identified 110 children treated for a B-lineage ALL harbouring a t(1;19(q23;p13) and/or E2A-PBX1 fusion transcript from 1987 to 2004 in the FRALLE protocols (FRALLE 83: n= 3, FRALLE 87–89: n=18, FRALLE 92 (phase II): n=10, FRALLE 93: n=48, FRALLE 2000 (still opened: n=31). Analysed features included: male sex (50, 45%), age (median: 6.9y; Q1–Q3: 3.5–12), CNS disease (4, 3.5%), leucocytosis (median: 21.4 G/L; Q1–Q3: 10.8–53.4), D8 poor prednisone response (PPR) (5/79, 6%), M1 D21 marrow response (103, 94%), and CR (108, 98%). For an accurate comparison, the subgroup of pts with t(1;19) included in the FRALLE 93 protocol (n=48) was compared to the 1,147 children aged more than 1 y with B- lineage ALL treated in the same protocol. Significant differences in the t(1;19) cohort were: excess of girls (65% vs. 46%; p= .01), and older age (median: 7 vs. 4.7; p= .01)). No differences in early response to treatment (D8, D21, D35) were seen. All the 110 patients received chemotherapy only, except 3 who received an autologous BMT for D8 PPR as recommended by the FRALLE 93 protocol. At a median FU of 65 months, 18 events have been recorded including 17 relapses (bone marrow: 13, CNS: 4) and 1 secondary tumor (abdominal Burkitt lymphoma) giving a 5 y and 10 y EFS of 78.7%. If “modern era” only is considered (> 1992), 5 y and 10 y EFS is 85.5%. Relapses occurred at a median time of 422 days (range: 72–899) in the whole t(1;19) cohort. In those from FRALLE 93, the median time to relapse (MTTR) was 280 days, that is significantly lower than the one observed in the control group defined above (MTTR = 894 days, Wilcoxon test, p = .01).

Conclusions: We confirm that a high cure rate is now associated to that subgroup of childhood ALL. The main finding is that no event except from the secondary Burkitt lymphoma (1705 d) was registered after 30 months from diagnosis in that cohort of 110 children with t(1;19)/E2A-PBX1 translocation associated ALL. It is then conceivable to announce cure at 36 months in that subgroup with an extremely low possibility of mistake, thus much earlier and with more confidence than in the other B-lineage ALL.
CD10− Pre-B Acute Lymphoblastic Leukemia (ALL): A Distinct High-Risk Subgroup of Adult ALL.
Authors: Gleissner Beate , Goekbuget Nicola , Rieder Harald , Hoelzer Dieter , Thiel Eckhard
Published: April 2020

Introduction: Immunological subtyping and molecular genetic analysis enable risk-adapted treatment of acute lymphoblastic leukemia (ALL). Translocations involving the mixed lingeage leukemia (MLL) gene characterize one poor-prognosis subtype of adult B-lineage precursor ALL correlating with a CD19+/CD10−/cytoplasmic (cy) IgM− immunophenotype (pro-B ALL).

Patients and Methods: Immunophenotyping was performed on 2,408 newly diagnosed ALL specimens indentifying pro-B ALL, TdT+/CD19+/CD10−/cyIgM−/surface(S)Ig−; c-ALL, TdT+/CD19+/CD10+/cyIgM−/SIg−; and pre-B ALL, TdT+/CD19+/CD10+/cyIgM+/SIg−. Ambiguous cases of CD10-positivity (5–15% CD10-positivity) were rechecked by a second antibody. BCR-ABL RT-PCR and cytogenetics were performed. CD10-negative blasts also underwent MLL-AF4 RT-PCR. FISH using directly labelled probes for MLL was done in selected cases. Patients were treated according the German Multicenter Adult ALL trials (GMALL) with MLL-rearrangement positive patients treated in the high-risk arm.

Results: Immunophenotyping identified 70 CD10-negative pre-B ALL specimens (TdT+CD19+cyIgM+). Fifty-six of these 70 CD10-negative pre-B ALL specimens contained a sufficient amount of blasts for molecular analysis. A total of 46/56 CD10-negative pre-B ALL were assigned to MLL-rearrangement positivity, revealing either MLL-AF4 fusion by RT-PCR (n = 38) or an 11q23/MLL translocation by cytogenetics (n = 4) or FISH (n = 4).

Looking at clinical characteristics, MLL-rearrangement-positive patients were characterized by a higher median white blood cell count (P = .0002) and their blasts showed frequently neuroglial antigen 2 chondroitin sulfate proteoglycan (P = .02).

Forty CD10− pre-B ALL patients with molecular genetic and/or cytogenetic data were evaluable for the treatment response and outcome. A complete remission (CR) was achieved in 6 of 7 (86%) MLL-rearrangement-negative and in 27 of 33 (82%) MLL-rearrangement-positive patients (P = .06). CR was maintained by 2 of 6 (33.3%) MLL-rearrangement-negative and 7 of 27 (26%) MLL-rearrangement-positive patients. The probability of remission duration after 3 years was 0.37 (± 0.29 SE) in MLL-rearrangement-negative versus 0.28 (± 0.13 SE) in MLL-rearrangement-positive patients contributing essentially to the overall survival at 3 years after diagnosis of 0.15 (± 0.06 SE) in patients with CD10-negative pre-B ALL.

Conclusion: Our data identify CD10− cytoplasmic Ig-positive pre-B ALL as a rare (2.2%) but distinct immunosubtype of adult ALL that is characterized by a high MLL rearrangement rate and a worse outcome with conventional intensified treatment strategies.
Microarray Analyses in a Case-Control Cohort of T-ALL Samples Identifies Gene Signature of Potential Prognostic Significance.
Authors: Winter Stuart S. , Khawaja Hadya , Jiang Zeyu , Kooperberg Charles , Ferrando Adolfo , Look Thomas , Larson Richard S.
Published: April 2020

Risk stratification remains limited for patients being treated for T-ALL due to a lack of biologic predictors of outcome. As a consequence, treatment assignment on modern protocols has been largely achieved through random assignment. Recent observations have suggested that overexpression of specific gene(s) may provide a reliable means to risk stratify patients. We hypothesized that microarray analysis may identify gene sets that distinguish both therapeutic response and patient outcome in T-ALL. We analyzed the gene expression profiles of 45 primary T-ALL samples (24 CCR, 21 relapse) from a matched, case control study with sufficient cRNA for microarray analysis (COG #8704). We performed oligonucleotide microarray analysis using Affymetrix U133Av.2 genechips which have approximately 54,000 target genes and ESTs. Following heirarchical clustering in dChip and R Language analyses (Chiaretti et al. Blood, 2004), but using RMA normalization, we identified 37 genes that serve as reliable predictors of CCR or relapse. Leave-one-out least discriminant analysis cross-over validation further constrained our prognostic gene identifiers to 21 genes of robust significance. These 21 genes predict 87 % of CCR and 82% of relapse accurately (p<0.0001, two-tailed Fisher’s exact test). These results were verified by qRT-PCR. Transcriptional factors previously described as having prognostic significance were not identified in our study. Twenty-six of the 45 cases received high dose L-asparaginase (16 CCR, 10 relapse) on the companion study. As a result, we examined whether a distinct signature could be also identified that distinguishes response to dose-intensified asparaginase treatment for patients with T-ALL. Using the same approach, a 27-member gene signature was identified that accurately predicted response in 24 of the 26 cases (92 %; 2 cases of relapse were misclassified). These results have identified two sets of genes that may be further pursued as prognostic indicators in T-ALL or as predictors of response to therapy.
Molecular Monitoring of Residual Disease (MRD) during Induction and Intensification Phases in Low Risk Adult B Cell ALL Treated According to the MRC UKALL12 Protocol.
Authors: Foroni Letizia , Mitchell Wayne , Rai Lena J. , Casanova Anouska , Gerrard Gareth , Patel Bella , Richards Sue , Fielding Adele , Goldstone Anthony H.
Published: April 2020

Introduction and aims. Molecular monitoring of minimal residual disease (MRD) using Immunoglobulin (IG) and T cell receptor (TCR) targets has provided an independent and prognostically significant parameter of outcome in adult and childhood acute lymphoblastic leukaemia (ALL). The aim of our study was to evaluate the impact of molecular tests carried out during the first 20 to 24 weeks of chemotherapy for MRD in a standard risk group of adult B cell ALL patients.

Patients and Methodology. We evaluated MRD tests in 63 patients with B cell ALL (37M/26F). Median age of our cohort was 24 yrs (range: 15.5–54.6 yrs) while median WBC was 9.9 (range: 1.1–553 × 109l). All were negative for the t(9;22) or t(4;11) translocation and received standard induction chemotherapy or auto stem cell transplant (A-SCT) only. All patients had at least one molecular marker which was tested by quantitative or semi-quantitative PCR with sensitivity ≥1E4 and all tests were carried out at time of morphological remission. End-points were relapse or continuous clinical remission (CCR) with follow up ≥12 months. Time point for molecular evaluation were post induction phase 1, TP1 (up to 1.8 mo), post induction phase 2 (1.8–3.5 mo), post intensification (3.5–5.7 mo). Median follow up was 92 months for patients in CCR (range 12–141 mo) and 15.5 months for patients who relapsed (range: 2.4–49 mo).

Results. Thirty-five patients in complete morphological remission were tested at TP1. Relapse free survival (RFS) tests showed a statistically significant correlation (p=0.02) between MRD positive tests and relapse (n=19 pts) and MRD negative tests (n=16 pts) and relapse free survival. In addition, pts with resistant disease at TP1 (n=10; not included above) and MRD positive pts were indistinguishable as far as relapse FS and both faired extremely poorly (Figure A).

Thirty patients were analysed at TP2. RFS confirmed the significant association between MRD positivity and poor outcome (n=16 pts) and MRD negativity and CCR (n=14 pts) (p=0.03) (Figure B).

MRD data were also available for 43 patients at TP3. Association of MRD positive tests (n=15 pts) and MRD negative tests (n=28 pts) with poor and good outcome, respectively (p=0.0006) (Figure C) was strongest at this time point. Outcome correlated with level of MRD at all time points, with poorer outcome in patients with MRD >1E3.

Conclusions. Molecular monitoring during induction and intensification for standard risk B cell ALL patients treated with UKALL12 protocol provides a prognostically significant parameter for the management of adult ALL in otherwise morphological remission and may in the future be used for patients’ stratification.

Figure Figure
The Value of Molecular Monitoring for Residual Disease (MRD) in Early Morphological Remitters among Adults Diagnosed with B Cell ALL and Treated According to the MRC UKALL12 Protocol.
Authors: Foroni Letizia , Mitchell Wayne , Rai Lena J. , Richards Sue , Fielding Adele , Goldstone Anthony H.
Published: April 2020

Molecular monitoring of minimal residual disease (MRD) has become an independent and prognostically significant parameter in assessing outcome in adult and childhood acute lymphoblastic leukaemia (ALL). The aim of our study was to evaluate the impact of MRD in a standard risk group of adult B cell ALL patients who had achieved morphological remission following induction phase 1 therapy as part of the MRC UKALL12 protocol.

Patients and Methodology. MRD tests were evaluated in fourty-seven patients with adult B cell ALL. They were negative for the t(9;22) or t(4;11) translocation and had received chemotherapy based treatment or auto stem cell transplant (A-SCT) only. Median age was 23 yrs (range 15.5–54.6 yrs); median WBC was 9.6 (range 1.1–163×109/l) with a predominance of common (28 pts) and pre B ALL (12 pts).

All patients had at least one molecular marker which was tested by quantitative or semi-quantitative PCR with sensitivity ≥1E4. End points were either clinical relapse or disease free survival in complete remission with follow up ≥12 months. Time point for molecular evaluation were post induction phase 1, TP1 (median 0.9 mo; range: 0.6–1.8 mo) and post induction phase 2 (median 2.79 mo; range 1.8–3.5 mo).

Results. Thirty three pts were tested at TP1 following morphological remission. Relapse free survival (RFS) analysis showed a statistically significant association between MRD positive tests (in 16 pts) and relapse, and MRD negative tests (in 17 pts) and CCR (p=0.03) (Figure 1, left diagram).

Twenty five pts were analysed at TP2 following their early morphological remission. At this time point we observed the strongest association between MRD negative tests (in 13 pts) and CCR (in 10 pts) and MRD positive (12 pts) and relapse (in 11 pts) (p=0.01)(Figure 1, right diagram).

Conclusions. Molecular monitoring of MRD shows that even among early morphological remitters a group of MRD positive patients can be identified that have poor overall outcome and may benefit from tailored therapies. Molecular assessment of residual disease should be used to stratify treatment in future adult ALL trials.

Figure Figure
Targeting CD20 and CD22 with Rituximab in Combination with CMC-544 Results in Improved Anti-Tumor Activity Against Non-Hodgkin’s Lymphoma (NHL) Pre-Clinical Models.
Authors: Hernandez-Ilizaliturri Francisco J. , Devineni Suresh , Arora Sarika , Knight Joy , Czuczman Myron S.
Published: April 2020

CMC-544 is an IgG4 CD22-humanized targeted immunoconjugate of calicheamicin. Upon CD22 biding, CMC-544 internalizes inducing cell apoptosis in vitro/in vivo against NHL with minimal modulation of the immune system. In addition to direct cell-death, rituximab, anti-CD20 monoclonal antibody (mAb), can induce complement-dependent cytotoxicity (CDC) and antibody dependent cellular cytotoxicity (ADCC). Combination of mAbs directed against unique tumor-associated targets can potentially enhance anti-tumor activity. Objectives: To study the effects of targeting CD20 and CD22 using rituximab in combination with CMC-544. Material and Methods: For in vitro studies, a panel of NHL cell lines [Raji, SUDHL-4, SU-DHL-10, Ramos, and two rituximab resistant cell lines (RRCL), [2R and 4RH] were exposed to CMC-544 (0.4pg/ml to 0.4μg/ml) +/− Rituximab (10μg/ml) or appropriate controls for 24 or 48 hrs. DNA synthesis was quantified by [H3]Thymidine incorporation and apoptosis was detected by multiparameter flow cytometric analysis and Poly(ADP-ribose) polymerase (PARP) cleavage. For ADCC/CMC studies, 51Cr-labeled NHL cells were exposed to CMC-544 (0.4μg/ml) prior to treatment with rituximab (10mg/ml) and peripheral blood mononuclear cells (Effector: Target ratio 40:1) or human serum, respectively. 51Cr-release was measured and the percentage of lysis was calculated. Statistical analysis of results was performed using the Chi-square test. For in vivo studies, 6–8-week old SCID mice were inoculated via tail vein injection (iv) with Raji cells (1 × 106 on day 0). Animals were assigned to receive 8 doses of either saline, CMC-544 (40μg/kg/dose), rituximab (10mg/kg/dose iv), or rituximab alternating with CMC-544. Antibodies were administered iv every other day starting on day +5. Survival analysis was performed by Kaplan-Meier curves and p values calculated using log rank test. Results: Exposure of various NHL cells to CMC-544 resulted in a dose-dependent significant decrease in cell proliferation and apoptosis. Rituximab-associated CMC or ADCC was not further enhanced by CMC-544 in vitro. In vivo studies demonstrated that the mean survival for animals treated with CMC-544 increased when compared to control mice [64 days (95% C.I. 48–80; 22 days (95% C.I. 21–23), respectively, P=0.001]. To a lesser degree rituximab monotherapy resulted in significant anti-tumor effects and the median survival reached 44 days (95% C.I. 31–56). Notably, animals treated with rituximab in combination with CMC-544 had the longest median survival (not reached at +90 days, log rank P=0.007). Conclusions: Our data suggest targeting CD20 and CD22 by rituximab and CMC-544 resulted in a longer survival in lymphoma-bearing SCID mice. Clinical trials with this combination will be needed to confirm these results for the treatment of lymphoma patients
Anti-Tumor Effect of Direct Intratumor Injection of Recombinant Fowlpox Virus Encoding CD40 Ligand in B Cell Lymphoma Model.
Authors: Liu Aichun , Guardino Alice , Panicadi Dennis , Chinsangaram Jarasvech , Levy Ronald
Published: April 2020

The interaction between CD40 and CD40 ligand (CD40L) on antigen presenting cells (APC) is critical in promoting cellular immune responses. In vitro experiments have shown that lymphoma B cells are efficiently infected by virus vectors encoding CD40L and subsequently up-regulate co-stimulatory molecules. By infection with CD40L, lymphoma B cells could function as APC. Tumor cells modified ex vivo to express CD40L have been shown to be able to induce protective immunity against subsequent tumor challenge. In order to develop an immunotherapy approach against established tumor, we tested the effect of intratumor injection of the recombinant fowlpox virus encoding murine CD40L (rF-mCD40L) together with systemic administration of cyclophosphamide (CTX). Methods: BALB/C mice were first inoculated s.c. with 1×107 syngeneic A20 lymphoma cells. On day 15, when tumor reached approximately 1.0 cm in diameter, mice were separated into 6 groups of ten mice which received: 1) rF-mCD40L alone, 2) empty fowlpox virus (FP-virus) alone, 3) PBS, 4) CTX (100mg/kg, i.p), 5) rF-mCD40L plus CTX, 6) FP-virus plus CTX. Virus was administrated twice by intratumoral injection at a dose of 1×109 pfu at a 7 days interval. CTX was delivered i.p at a dose of 100mg/kg on two successive days between the two virus injections. Tumor growth and animal survival were monitored every three days. In addition, tumor cell transgene expression was measured by flow cytometry 24 hours following virus injection. Results: After 24 hours of intratumor injection, a small percentage of tumor cells shown to be positive for the transgene on analysis. As shown in Figure 1, Chemotherapy alone caused only temporary regression of tumor, but the combination of rF-mCD40L with CTX resulted in tumor free mice 80 days after tumor inoculation. By contrast, mice treated with rF-mCD40L alone or with empty virus, died 4 to 7 weeks after tumor inoculation. 40 to 50% of mice treated with CTX alone or with FP virus combined with CTX exhibited re-occurrence of tumor growth and 3 mice in group of CTX alone, 2 mice in the group of FP virus combined with CTX died of tumor progression. Conclusion: rF-mCD40L alone had no anti-tumor effect against established lymphoma. Intratumor injection of rF-mCD40L in combination with systemic chemotherapy was effective for the therapy of advanced lymphoma. Intratumor administration of CD40L in combination with chemotherapy may be a promising strategy for the treatment of advanced lymphoma.

Figure Figure
The PKCβ Selective Inhibitor, Enzastaurin (LY317615), Inhibits Growth of Human Lymphoma Cells.
Authors: Rossi Randall M. , Henn Alicia D. , Conkling Rebecca , Guzman Monica L. , Bushnell Timothy , Harvey Jennifer , Fisher Richard I. , Jordan Craig T.
Published: April 2020

Activation of PKC contributes to tumor cell survival and proliferation and has been implicated in the pathogenesis of human B cell lymphomas. Specifically, PKCβ activation is increased in tumor cells from patients with poor prognosis diffuse large cell lymphoma (DLCL), suggesting that PKCβ may be a target for therapeutic intervention. In this study we tested the PKCβ inhibitor, Enzastaurin (LY317615), using both cell culture and xenograft assays, on a panel of eight DLCL lines (OCI-Ly3, OCI-Ly7, OCI-Ly10, OCI-Ly19, SUDHL-4, SUDHL-6, Farage, and Toledo). In suspension culture, Enzastaurin was cytotoxic to all lines tested, with an average IC50 at 48 hours of 2.0 micromolar (range 0.5–4.0 micromolar). Molecular analyses showed that Enzastaurin treatment inhibited phosphorylation of GSK3β, a potential pharmacodynamic marker of Enzastaurin activity. Subsequent studies were performed using two subclones of the DLCL lines OCI-Ly10 and OCI-ly19, which respectively represent the ABC (activated B cell) and GC (germinal center) molecular subclasses of DLCL. Each line was engineered to express luciferase, which permits real time in vivo imaging. Upon subcutaneous transplantation into immune deficient NOD/SCID mice, both lines formed reproducible tumors. Treatment with Enzastaurin (75mg/kg, b.i.d. oral gavage) was initiated during the first 1–2 weeks post-transplant, before palpable tumors were evident, and was continued for 14 consecutive days. The Enzastaurin treatment was well tolerated, with the only overt side effect being moderate weight loss. At the end of therapy, vehicle control treated tumors had become palpable and progressed to volumes of 40–60 cubic millimeters. In contrast, Enzastaurin treated tumors (both OCI-Ly10 and OCI-Ly19) showed no observable progression, remaining detectable by luciferase imaging, but non-palpable. Collectively, these data indicate the Enzastaurin can induce death of human DLCL lines in vitro and is at least cytostatic to human DLCL in vivo.
Targeting Lymphoid Malignancies with Small Molecule Inhibitors of Pro-Survival Bcl-2 Proteins.
Authors: Roberts Andrew W. , Wei Andrew , van Delft Mark F. , Mason Kylie D. , Adams Jerry M. , Huang David C.S.
Published: April 2020

As overactivity of pro-survival Bcl-2 proteins promotes neoplasia and enhances resistance of malignant cells to cytotoxic therapies, a promising approach for incurable lymphoid tumors is to directly target the pro-survival proteins. One approach is to mimic their physiological antagonists, the BH3-only proteins. We initially determined which of the diverse BH3-only proteins would be optimal to mimic. The interactions of the BH3 domains of this family with a groove on the Bcl-2-like proteins have been considered promiscuous. However, we found that the interactions between eight BH3 peptides and five Bcl-2-like proteins varied over 10,000 fold in affinity, and that only certain protein pairs associate inside cells (Chen et al Mol Cell;17:393–403, 2005). Bim and Puma potently engaged all the pro-survival proteins comparably. Bad, however, bound preferentially to Bcl-2, Bcl-xL and Bcl-w. Strikingly, Noxa bound only Mcl-1 and A1. In accord with their complementary binding, Bad and Noxa cooperated to induce potent killing.

We next determined the mechanism of action of putative BH3 mimetic compounds. Because killing by the BH3-only proteins require the action of the essential cell death mediators, Bax and Bak, we initially screened putative BH3 mimetic compounds on cells genetically engineered to lack both Bax and Bak. Only those compounds that mimicked the BH3-only proteins (i.e, inert on BaxBak double null cells, but active in Bax or Bak expressing cells) were evaluated further. Surprisingly, among seven putative BH3 mimetics tested, we found that only the recently described ABT-737 (Abbott Laboratories; Oltersdorf et al, Nature435:677–81, 2005), required the pro-apoptotic protein Bax or Bak to induce apoptosis. The cytotoxicity of ABT-737 alone was modest in a range of hematopoietic and non-hematopoietic cell types. Further investigations revealed that, like Bad, ABT-737 only targeted Bcl-2, Bcl-xL and Bcl-w. As hematopoietic cells typically express Mcl-1, we attempted to augment the activity of ABT-737 by concomitantly neutralizing Mcl-1. Targeting of Mcl-1 by overexpressing the BH3-only protein Noxa, or Mcl-1 down-regulation by RNAi, cytokine deprivation or cytotoxic agents, allowed ABT-737 to efficiently kill diverse cell types, even when Bcl-2 was over-expressed. We conclude that ABT-737 is a highly selective and specific BH3 mimetic compound that should prove highly efficacious in tumors where Mcl-1 is low, or when combined with agents that down-regulate Mcl-1. This hypothesis is currently being tested in vitro in primary human lymphoid malignancies, and in vivo using immunocompetent murine models of lymphoma. ABT-737 also provides strong proof-of-principle that targeting pro-survival Bcl-2 proteins is feasible, but the optimal utility of such BH3 mimetics depends on a thorough understanding of the pathways to apoptosis.
Combination of Farnesyl Transferase Inhibitor (Tipifarnib) with Perifosine Induces Apoptosis through phos-PDK1 in Human Lymphoma and Leukemia Cell Lines.
Authors: David Ebenezer , Sinha Rajni , Torre Claire , Kaufman Jonathan L. , Lonial Sagar
Published: April 2020

Introduction: Novel agents as anti-cancer therapy are used in the setting of specific molecular abnormalities that provide a survival advantage for malignant cells. One such agent, tipifarnib, is theoretically targeted at Ras mutations which are present in a number of different human cancers. Our previous experience with the FTIs (David et al, in press Blood) has demonstrated that they are ideal agents to combine with other targeted agents. We have investigated the combination of the AKT inhibitor perifosine with tipifarnib in human leukemia and lymphoma cell lines with the hypothesis that the combination of 2 targeted agents will disrupt separate survival pathways and ultimately result in synergistic tumor cell death.

Methods: In this study we used the human leukemia cell lines HL-60, Jurkat, and the lymphoma cell line HT. Western blot analysis was used to assess for the effect of either single agent perifosine, tipifarnib, or the combination on AKT, p-AKT, PDK-1, and caspase cleavage. Flow cytometry was utilized to assess for Annexin V staining following combination therapy.

Results:Dose escalation studies demonstrated that doses of tipifarnib up to 5μm demonstrated a significant cell death in HL-60 and HT cells. Perifosine doses of 1–5uM also induced cell death in both HL-60 and HT cells. When apoptosis was assessed using western blot analysis of caspase 3 activity and cleavage, the combination of perifosine and tipifarnib demonstrated significant apoptosis using low doses of both agents. The apoptosis was associated with downregulation of phos-PDK1, with a resultant downregulation in p-AKT. The level of phos-PDK1 was completely inhibited in less than 24 hrs in both the HL-60 and HT cell lines in combination than when either agent was given alone.

Conclusion: The combination of perifosine, and AKT targeted agent, with tipifarnib, a Ras targeted agent, appear to induce significant cell death in lymphoma and leukemia cell lines with rapid downregulation of p-AKT via the PDK-1 pathway. This apoptosis occurs in vitro using concentrations well below those that have been achieved in current clinical trials using these agents. Additional studies are being carried out to further delineate the mechanism of synergy as well as to further explore the impact of sequence of administration using this combination. Further studies are also planned to xplore the impact of the combination on primary human leukemia and lymphoma cells from the blood and bone marrow.
The Small Molecule Pan-Bcl-2 Inhibitor GX15-070 Induces Apoptosis In Vitro in Mantle Cell Lymphoma (MCL) Cells and Exhibits a Synergistic Effect in Combination with the Proteasome Inhibitor Bortezomib.
Authors: Galan Patricia Perez , Roue Gael , Villamor Neus , Campo Elias , Colomer Dolors
Published: April 2020

Mantle cell lymphoma (MCL) is a lymphoid malignancy derived from a subset of mature B-cells with coexpression of CD5 and the chromosomal translocation t(11;14)(q13;q32). MCL patients present an aggressive clinical course and poor response to conventional chemotherapy due to either rapid relapse after an initial response or primary resistance to drugs. Thus, there is currently a strong effort to develop compounds that target novel biological pathways. In addition, MCL cells overexpress the antiapoptotic proteins Bcl-2, Bcl-XL and Mcl-1. The dysregulated Bcl-2 pathway represents an important target for a new-mechanism therapeutic approach. In this context, the pan-Bcl-2 inhibitor GX15-070 that belongs to a new family of compounds which mimic BH3-only proteins by binding to multiple antiapoptotic Bcl-2 members and consequently eliciting Bax and Bak dependent cytotoxicity. GX15-070 is currently being evaluated in Phase I clinical trials. GX15-070 induced apoptosis in vitro in primary cells from MCL patients as well in MCL cells lines, at doses ranging from 0,5μM to 5μM. GX15-070 induced phosphatidylserine exposure, mitochondrial depolarization, ROS generation, Bax and Bak conformational changes and caspase activation. As expected, pan-caspase inhibitors did not block apoptosis in accordance with the mitochondria-targeted nature of this new compound. The susceptibility of MCL cells correlates with the expression of Bcl-2, Mcl-1 and Bcl-XL, both at protein and mRNA levels. Although GX15-070 exhibited significant in vitro cytotoxicity in MCL as a single agent, we tested also this compound in combination with proteasome inhibitors. Bortezomib (PS-341, Velcade®) has shown promising results in MCL treatment. One of the drawbacks of proteasome inhibitors is the undesirable accumulation, due to absence of degradation, of antiapoptotic factors which could impede or slow apoptosis, such as Mcl-1 protein. With this rationale we evaluated a combination of bortezomib with the pan-Bcl-2 inhibitor GX15-070 in vitro in MCL cells. Preincubation of MCL cells with GX15-070 at doses ranging from 0,1μM to 1μM before bortezomib (1–10nM) addition induced a synergistic cytotoxic effect (Chou Talalay CI =[0,26–0,575]). In vitro, this new combination allows to reduce bortezomib dose 5 to 20 times, depending on the individuals. Western blot analysis revealed that this combination reduced Mcl-1 levels and displaced Bak from Mcl-1 sequestration. Moreover, upregulation of the proapoptotic BH3-only protein Noxa by bortezomib, was further increased when combined with GX15-070. All these findings suggest that GX15-070 and bortezomib combination could be a new therapeutic approach for MCL treatment.
Dose Escalated CHOP + Etoposide Followed by Repetitive Autologous Stem Cell Transplantation (MegaCHOEP) with or without Rituximab for Primary Treatment of Aggressive NHL.
Authors: Glass Bertram , Kloess Marita , Reiser Marcel , Metzner Bernd , Truemper Lorenz H. , Loeffler Markus , Pfreundschuh Michael , Schmitz Norbert
Published: April 2020

Whilst safety and efficacy of adding rituximab to standard therapy has been reported, less information is available on the combination of rituximab (R) with HDT and autologous SCT. The German Study Group for High-Grade NHL (DSHNHL) developed a repetitive HDT program consisting of 4 courses dose escalated CHOP + Etoposide +/− R. Patients aged 18 to 60 years and elevated LDH at diagnosis were included. Mega-CHOEP consisted of four cycles of chemotherapy. Cycles 2 to 4 were followed by autologous SCT. At dose level 3 (DL3) cumulative doses of drugs were as follows: C 19.5 g/m2, H 280 mg/m2, O 8 mg, E 5.04 g/m2, P 2000 mg. When feasibility and safety of MegaCHOEP at DL3 had been demonstrated, rituximab was added (DL3R). Six cycles of rituximab (375 mg/m2) were given: one dose of rituximab prior to each cycle of chemotherapy and two additional doses 3 and 6 weeks after the last cycle of chemotherapy, respectively. From September 1999 to may 2001 35 pts with validated diagnosis of aggressive B-NHL received MegaCHOEP DL3, from June 2001 to February 2003, 72 pts received DL3R. There was a higher percentage of B-cell NHL patients with ECOG performance status > 1 at DL3 (42.9 %) than at DL3R (19.7 %, p = 0.012) and as consequence more patients with age-adjusted IPI high risk at DL3 (34.3 %) than at DL3R (12.7 %, p = 0.029). We observed significant differences in the incidence of WHO grade III/IV infections (DL3 9 of 123 cycles (7.3%), DL3R 48 of 236 cycles (20.3%); p=0.001) and in the percentage of patients receiving the complete program of chemotherapy (DL3:91 %, DL3R 71%; p=0.017). There were no relevant differences in other types of non-hematologic toxicity or hematopoietic recovery between treatment groups. Median observation times of patients at DL3 and DL3R were 2.6 years and 4.5 years, respectively. In univariate analysis, there was a trend to better overall survival with rituximab (75%) than without rituximab (57%) at three years (p=0.168) and a significantly better freedom from treatment failure in patients receiving DL3R as compared to DL3 with 70 % vs 50 % at 3 years (p = 0.040). In a Cox regression model adjusted for IPI, there was a trend to a higher relative risk of treatment failure of MegaCHOEP without rituximab (RR 1.8); this, however, was not statistically significant (p = 0.087). Rituximab may have beneficial influence on lymphoma control in this setting; its use, however, is accompanied by higher incidence of serious infections which subsequently compromise the dose intensity of this aggressive treatment program. The DSHNHL is currently performing a randomized study comparing MegaCHOEP+R with CHOEP−14+R.
Rituximab in Relapsed Lymphocyte Predominant Hodgkin’s Disease (LPHD). Long-Term Results of a Phase-II Study from the German Hodgkin Lymphoma Study Group (GHSG).
Authors: Schulz Holger , Trelle Sven , Reiser Marcel , Sieber Marcus , Diehl Volker , Engert Andreas
Published: April 2020

Introduction: LPHD is a rare disease which accounts for 3–8% of all Hodgkin cases. Patients with LPHD relapse frequently and freedom from treatment failure is not significantly improved by intensification of polychemotherapy or radiotherapy. The malignant cells of LPHD are CD20+ and therefore the anti-CD20 antibody rituximab (R) may have activity with fewer adverse late effects.

Methods: This phase-II trial was initiated by the German Hodgkin Lymphoma Study Group (GHSG) to evaluate rituximab in patients with LPHD at first or higher relapse or progressive disease after at least one standard treatment. Histological slides were reviewed by a reference panel. Pts received 375mg/m2 of the anti-CD20 antibody rituximab once weekly for four weeks given as intravenous infusion in saline solution.

Results: Between 1999 and 2004 we treated twenty-one pts. with CD20-positive Hodgkin’s lymphoma according to the study protocol. Fourteen patients had stage I/II disease at the time of study entry and all patients were in their first to third relapse (median 2). The initial diagnosis of LPHD was confirmed in 17/21 cases. The remaining cases were reclassified as HD transformed to T-cell rich B-cell lymphoma (2) or CD20-positive classical HD (2). The overall response rate was 90%. Time to progression was 31 months (ms). The median follow-up 58 ms. Both T-cell rich B-cell lymphoma are in continous remission (PR 51ms+, CR 61ms+).

Conclusion: Single-agent therapy with rituximab is safe and showed high efficacy in relapsed LPHD. Therefore rituximab might be a non-toxic and efficient alternative treatment strategy compared to intensified chemo-and/or radiotherapeutic protocols in this young group of patients.
Adoptive Immunotherapy with Autologous CD25-Depleted and CD3/CD28-Costimulated T-Cells (ACTC) Enhances Numeric and Functional Lymphocyte Recovery after Cyclophosphamide - Fludarabine Chemotherapy in Patients with Low-Grade Follicular Lymphoma.
Authors: Andreadis Charalambos , Levine Bruce L. , Cotte Julio , Zheng Zhaohui , Mair Rebecca , Cannon Andrea , Harrow Jason , Traum Marcy , Chong Elise A. , Downs Lisa H. , Vogl Dan T. , Svoboda Jakub , June Carl H. , Schuster Stephen J.
Published: April 2020

Fludarabine-based chemotherapy combinations are highly effective in patients (pts) with low-grade follicular lymphoma (FL), but cause severe and long-lasting immunosuppression due to depletion of normal CD4 T-cells. Aside from increasing the risk of serious infections, this toxicity may limit the ability of the immune system to eliminate minimal residual disease. Adoptive immunotherapy using autologous CD25-depleted, CD3/CD28-costimulated T-cells expanded ex vivo (ACTC) may enhance immune reconstitution and improve disease control. We initiated a phase I study in pts with purine analog-naive relapsed/refractory FL (grades 1 and 2). After leukapheresis, pts receive 4 cycles of fludarabine (25 mg/m2) days 1–3 and cyclophosphamide (250 mg/m2) days 1–3. Four weeks after last chemotherapy, responding patients (CR, CRu, PR) receive escalating doses of ACTC prepared ex vivo from autologous T-cells collected prior to chemotherapy and depleted of regulatory CD4+/CD25+ cells, then expanded and activated using anti-CD3 and anti-CD28. Eight pts have been enrolled to date. Median age is 40.5 y (range: 32–64). Median number of prior therapies is 2 (range: 1–3). Two pts were withdrawn from the study due to hematologic toxicity related to the chemotherapy; one patient has not completed chemotherapy. Of the 5 pts completing chemotherapy, 3 pts achieved a CR and 2 pts achieved a PR; 4 pts received 5 x 109 and 1 patient received 1 x 1010 CD3+ ACTC. There have been no adverse events related to T-cell infusions. Median follow-up after ACTC infusion is 9 months (range: 2–15 months). Median time to CD4 count >200 /uL was 29 days following T-cell infusion (range 28–127 days). CD4 counts increased in all patients by 1 month after T-cell infusion, with a median increase of 126% from baseline (range 50 to 484%). CD8 counts also increased, with a median increase of 82% (range −4 to 976%). All 5 pts receiving ACTC were anergic to candida antigen by Delayed Type Hypersensitivity (DTH) skin testing before chemotherapy. Three pts developed a positive DTH response to candida antigen 60 days after ACTC infusion. From the start of therapy for patients receiving T-cells, median follow-up is 14 months (range 7–20 months) with median progression-free survival not reached; 4 pts remain in remission and 1 patient had progression of disease. ACTC results in significant CD4+ lymphocyte recovery in previously treated pts receiving cyclophosphamide-fludarabine chemotherapy and compares very favorably with historical controls. T-cell dose escalation is ongoing.