bluuebunny/crossref_metadata_2025 huggingface dataset:
Murine NK Cells Require Activation-Dependent Expression of Granzyme B and Perforin To Become Potent Cytotoxic Effectors.
Authors: Fehniger Todd A. , Cai Sheng F. , Cao Xuefang , Bredemeyer Andrew J. , Presti Rachel M. , French Anthony R. , Ley Timothy James
Published: April 2020

NK cells predominantly utilize the granule exocytosis pathway to kill virus-infected and malignant target cells. Current paradigms suggest that resting NK cells have pre-formed granules containing granzymes A, B, and perforin and are ready to kill targets immediately upon proper recognition by NK receptors. Here, we report that resting murine NK cells in the spleen exhibit poor cytotoxicity (5.4±1.6% target cell death, 20:1 E:T ratio and 4 hour incubation), compared with cytokine-activated (IL-15, 48 hours) splenic NK cells (59.7±10.6% target cell death), against the RMAS tumor cell line in vitro as measured by a flow-based killing assay. In addition, using intracellular flow cytometric analysis with monoclonal antibodies specific for granzymes A, B, and perforin, we find that resting murine NK cells express abundant granzyme A (86.2±1.9% positive), but little or no granzyme B (4.4±5.4% positive) or perforin (2.6±1.8% positive). Activation of murine NK cells with IL-15 induces robust expression of both perforin (59.1±2.0% positive) and granzyme B (91.5±7.9% positive), which correlates with increased cytotoxicity. Further, granzyme B cluster −/− (26±6.7% target cell death) and perforin −/− (5.7±1.3% target cell death) NK cells have poor cytotoxicity in vitro despite IL-15 activation. Poly I:C simulates RNA virus infection and activates NK cell cytotoxicity in vivo through TLR3 and cytokine cascades. NK cell granzyme B and perforin expression is induced in vivo 24 hours after poly I:C injection, correlating with increased in vitro NK killing of tumor targets. In wild type mice infected with murine cytomegalovirus (MCMV), NK cell expression of both perforin (83.5±4.9% positive) and granzyme B (89.3±2.1% positive) is upregulated in the spleen, peaking 2–4 days post-infection and returning to baseline by 8 days post-infection. In addition, MCMV titers are significantly elevated at day 3 post-infection in both granzyme B cluster −/− (P<0.01) and perforin −/− (P<0.01) mice, compared to wild type mice. Moreover, survival following MCMV infection was significantly lower in granzyme B cluster −/− and perforin −/− mice, compared with wild type mice (P<0.001, see survival curve). Thus, our findings show that murine NK cells require the activation of granzyme B and perforin to become potent cytotoxic effectors. We also demonstrate for the first time that granzyme B is critical for early host defense against MCMV. These findings explain the long-standing observation that murine NK cells require prior activation for potent natural killing of tumor targets in vitro. Further, this requirement for activation-dependent granzyme B and perforin expression in NK cells may influence outcomes in murine models of innate immune anti-tumor and anti-viral responses.

Figure Figure
Down-Regulation of Glucocorticoid-Induced TNF Receptor (GITR) Ligand on Human Tumors by Proteolytic Shedding Increases Anti-Tumor Reactivity of NK Cells.
Authors: Baltz Katrin M. , Krusch Matthias , Baessler Tina , Kloss Mercedes , Kumbier Ingrid , Peterfi Andrea , Kanz Lothar , Salih Helmut R.
Published: April 2020

Members of the TNF superfamily mediate multiple cellular functions, including cellular proliferation, differentiation and cell death. Many members of this protein family are shed from the cell surface as soluble forms, which affects cell-cell interactions by reduction of ligand densities and distally modulates effector cells bearing the respective receptor. The TNF family member Glucocorticoid-induced TNF Receptor (GITR) costimulates effector T cells, modulates apoptosis and NFkappaB and abrogates suppression of murine but not human regulatory T cells. Its cognate ligand GITRL has been found in various healthy tissues. Recently we reported that NK cells express GITR, while tumor cells express GITR ligand (GITRL), and GITR/GITRL interaction downregulates NK cell-mediated anti-tumor effector mechanisms like cytotoxicity and IFN-gamma production. Here we report that human tumor cells spontaneously release a soluble form of GITRL (sGITRL) detectable in culture supernatants by ELISA. Furthermore, we found elevated levels of sGITRL in sera of patients with various malignancies compared to healthy controls. We demonstrate that the release of GITRL from tumor cells can be blocked by inhibition of metalloproteinases, concomitantly causing accumulation of GITRL on the tumor cell surface as determined by FACS analysis. Upregulated GITRL surface expression further increased inhibition of NK cell anti-tumor effector mechanisms, while, in contrast, presence of sGITRL in cocultures of GITRL-expressing tumor cells and GITR-positive NK cells enhanced NK cell cytotoxicity and IFN-gamma production. Thus, in line with the results obtained with other TNF family members, conversion of membrane bound GITRL to its soluble form is associated with downregulation of its function, potentially due to blocking its cognate receptor. Thus, release of sGITRL substantially influences the interaction of tumor cells with NK cells. In addition, determination of sGITRL levels may be implemented as a diagnostic marker in patients with malignancies. Further prospective studies are currently being conducted addressing the value of GITRL as a tumor marker in different tumor entities.
Regulation of Dendritic Cell Function by Histone Deacetylase Inhibitors.
Authors: Beck Julia , Nencioni Alessio , Grünebach Frank , Bringmann Anita , Kanz Lothar , Patrone Franco , Ballestrero Alberto , Brossart Peter
Published: April 2020

Histone deacetylases (HDACs) modulate gene transcription and chromatin assembly by modifying histones at the post-transcriptional level. HDAC inhibitors have promising antitumor activity and are presently explored in clinical studies for malignancies of different histology. Preliminary evidence in animal models of immune disorders also suggests immunosuppressive properties for these small molecules, although the underlying mechanisms remain at present poorly understood. Here, we show that two HDAC inhibitors of clinical use, sodium valproate and MS-275, profoundly affect cytokine-induced differentiation of human monocytes to dendritic cells (DCs), and impair DC phenotypic maturation in response to the Toll-like receptor (TLR) ligand polyinosinic:polycytidylic acid (poly I:C) characterized by a reduced expression of HLA- and costimulatory molecules. HDAC inhibitors reduce cytokine secretion important for T-cell activation like IL-12 and immunostimulatory capacity in DCs and inhibit DC migratory capacity toward the chemoattractant CCL19/MIP-3β. The observed defects in DC function upon exposure to HDAC inhibitors were not due to an increased rate of apoptosis or production of IL-10. We finally investigated the effects of HDAC inhibition on intracellular signalling pathways that play a key role in controlling DC differentiation and function. HDAC inhibitors have been reported to cause a downregulation of the hematopoietic transcription factor PU.1, which contributes to the development of thymic and myeloid dendritic cells and of Langerhans cells in the mouse. We evaluated herein whether PU.1 expression was affected by VPA in monocyte-derived DCs, however, no significant reduction in protein levels could be detected. Therefore, our data rule out PU.1 shortening as a mechanism responsible for the functional defects observed in DCs that were exposed to HDAC inhibitors. Similarly, no significant effect of HDAC inhibitors was detected on Bcl-6, another known target of HDAC inhibitors that, besides its role in lymphomas, has also been linked to the regulation of cytokine and chemokine release in macrophages. In contrast, addition of VPA blocked RelB nuclear relocalization in response to poly I:C in a concentration-dependent manner. Interestingly, IRF-3 and IRF-8, which have been involved in DC differentiation, IL-12 production and DC migration, were also affected by HDAC inhibitors, since their nuclear levels were reduced by VPA in unstimulated as well as in poly I:C-activated DCs. On the other hand, IRAK-1 downregulation in response to poly I:C was not affected by VPA, suggesting that the effects of HDAC inhibition on RelB, IRF-3 and IRF-8 are probably mediated downstream of MyD88 and IRAK-1.

In conclusion, HDAC inhibitors exhibit strong immunomodulatory properties on human DCs. Our results support the evaluation of HDAC inhibitors in inflammatory and autoimmune disorders.
Comparison of 7-Day Storage of Buffy-Coat Derived Platelet Concentrates in an Osmotically Balanced, Interface Stabilizing Platelet Storage Solution and in Plasma.
Authors: Zhang Jerry G. , Culibrk Brankica , Devine Dana D.V. , Hoang Lien , Hunter Faith , Kerr Ian , Levin Elena , Scammell Ken , Stewart Tamiko , Weiss Sandra , Gyongyossy-Issa Maria I.C.
Published: April 2020

The use of platelet additive solutions (PAS) for the storage of buffy-coat derived platelet concentrates (PC) could conserve plasma resources and reduce post-transfusion reactions. However, PASs are difficult to handle in a production environment where their relative low viscosity produces an unstable interface between the platelet rich plasma and the red cell pellet. In the current study, a comparison is drawn between buffy-coat derived PCs (4 buffy-coats per pool) stored over 7 days either in plasma or in a novel Viscous PAS that contains 26 % plasma as a by-product of the buffy-coat pooling process. The increased viscosity (1.18 cp at 37°C) of the PAS results in a stable interface during processing. Similar, though more consistent platelet recoveries from buffy-coat pools (BCP) are achieved by the Viscous PAS (77.0 ± 2.6%) as compared to plasma (73.7 ± 5.9 %). These are significantly higher (p<0.05) than recoveries using conventional PASs (64.5 ± 2.1 %). On day 7, platelet concentration, mean platelet volume (MPV), and soluble protein concentration in PCs stored using Viscous PAS (782 ± 87 x 109 cells/L, 9.1 ± 0.5fL, 15.0 ± 1.6 mg/L, respectively) and plasma (840 ± 104 x 109 cells/L, 9.0 ± 0.7fL, 56.09 ± 9.2 mg/L, respectively) were not significantly different from their corresponding day 1 parameters (837 ± 84 x 109 cells/L, 9.1 ± 0.3 fL, 13.5 ± 0.2 mg/L) respectively for Viscous PAS and (830 ± 73 x 109 cells/L, 8.3 ± 0.5 fL, 56.6 ± 6.5 mg/mL) and for plasma. This indicates that Viscous PAS and plasma maintain platelet integrity to a similar degree during 7-day storage. Both storage media maintain pH around 7.20 until day 7. However, the rate of glucose consumption and lactate production of PCs stored in Viscous PAS (0.3365 mmol/L.day, and 0.7591 mmol/L.day) was roughly half that of the rate exhibited by PCs stored in plasma (0.8447 mmol/L.day, and 1.4258 mmol/L.day). As lactate production and glucose consumption are correlated with in vivo platelet recovery (Goodrich et al., 2006) this augurs well for platelets stored in Viscous PAS. Monitoring the extent of shape change (ESC) and morphology scores for PCs in either of the storage media showed no significant difference on day 7. However, CD62 surface expression and the hypotonic shock response (HSR) were significantly better at day 7 (p<0.05) for platelets stored in the Viscous PAS (37.43 ± 3.31% and 75.76 ± 10.95%, respectively) than stored in plasma (58.37 ± 11.71% and 49.79 ± 15.34%, respectively). We have developed a PAS that is easy to handle in a production environment and because of its osmotic balance and realtive viscosity exhibits equivalent or better storage parameters over a 7 day period than does plasma.
High Definition Contrast-Enhanced MR Imaging in Paroxysmal Nocturnal Hemoglobinuria (PNH) Suggests a High Frequency of Subclinical Thrombosis.
Authors: Hill Anita , Reid Scott A. , Rother Russell P. , Gladwin Mark T. , Collinson Paul O. , Gaze David C. , Lowe Angela , Guthrie Ashley , Sivananthan Mohan U. , Hillmen Peter
Published: April 2020

Paroxysmal nocturnal hemoglobinuria (PNH) is an acquired disorder characterized by intravascular hemolysis and venous thrombosis. Thrombosis is the most feared complication in PNH and is reported to occur in >40% of patients. Proposed mechanisms of thrombosis include depletion of the coagulation regulator nitric oxide (NO) by intravascular hemolysis and increased sensitivity of PNH platelets to activation. The occurrence of subclinical thrombosis in PNH patients has not been previously studied using modern imaging techniques. In order to evaluate for subclincal thrombosis we evaluated PNH patients with a comprehensive state-of-the-art MRI protocol (which included the use of both blood pool and conventional Gadolinium based contrast agents) for the detection of subclinical thromboses and its sequelae. The detailed protocol consisted of:

lung perfusion and pulmonary MRA, cardiac MR - including quantitative studies of both ventricles, right heart flow dynamics and delayed enhancement for the detection of left ventricular damage, and abdominal MR for the assessment of hepatic and portal venous systems and kidneys.

10 PNH patients (median age 31.5 yrs) with large PNH clones but without previous clinical evidence of venous or arterial thrombosis underwent imaging. Five (50%) of the patients were on primary anticoagulant prophylaxis with warfarin. There was evidence of significant renal hemosiderosis, which was distributed throughout the cortices, in 8/10 patients. Two patients had small myocardial scars suggestive of previous unsuspected ischemic damage. Six patients had sub-segmental perfusion defects mainly distributed in the peripheries of the lung fields indicative of previous small pulmonary emboli. No such subclinical thromboses would be anticipated in an age-matched control population. 8 patients had mildly reduced right ventricular ejection fractions (mean 42.2±1.8%; normal range 48–63%). The plasma B-type natriuretic peptide (BNP) level was high in all 10 patients (median 29.4pmol/l; range 18.7–373.90; normal subjects 4.89±1.00pmol/l). BNP has been shown to increase in proportion to right ventricular dysfunction in pulmonary hypertenstion. No intraabdominal defects were identified with the current protocol. In summary, we identified abnormalities suggestive of previous subclinical thromboses in 6 of 10 hemolytic PNH patients by high-resolution MR imaging, including in patients on primary prophylaxis with warfarin. Effective prevention of thrombosis is an important aspect of the therapy in PNH.
A Prospective Trial of Radiation Alone vs Combination Chemotherapy Alone for Early-Stage Hodgkin’s Disease: Implications of 25-Year Follow-Up to Current Combined Modality Therapy.
Authors: Longo Dan L. , Glatstein Eli , Duffey Patricia L. , Young Robert C. , Fiem Shelby , Jaffe Elaine S. , Camphausen Kevin , Wilson Wyndham , DeVita Vincent T.
Published: April 2020

We conducted a prospective randomized trial comparing radiation therapy to MOPP chemotherapy in patients with stages IA through IIIA Hodgkin’s disease (HD). Patients with peripheral stage IA disease received radiation therapy without randomization. Median follow-up is now 25 years. Among the 30 patients with peripheral stage IA disease, all achieved complete response, 4 relapsed (13%), 8 died [27%; 2 from HD, 1 suicide, 1 myocardial infarction (MI), and 4 from second malignancies). Among the 51 patients randomized to receive radiation, 49 (96%) achieved complete response, 19 relapsed (39%), 18 died (35%; 10 from HD, 1 AIDS, 1 anorexia nervosa, 1 pulmonary fibrosis, 3 congestive heart failure, and 2 from second malignancies). In addition, 7 other second cancers have been observed in 6 patients treated with radiation who are alive and significant treatment toxicities affect the lives of 5 others. Among the 54 patients randomized to receive MOPP chemotherapy, 52 (96%) achieved complete response, 7 relapsed (13%), 10 died [18%; 5 from HD, 2 MI (1 in cycle 1), 1 from Pneumocystis pneumonia during treatment, and 2 from second malignancies]. Two other patients are alive with a second cancer and one has AIDS. Disease-free survival is 87% for MOPP and 61% for radiation in the randomized arms at 25 years (p=0.0034). Overall survival is 81% for MOPP and 63% for radiation in the randomized arms at 25 years (p=0.048). When the peripheral IA patients are added to the randomized radiation-treated patients, disease-free survival is 70% (p=0.36 vs MOPP) and overall survival is 67% (p=0.07 vs MOPP). The design of the study permits an analysis of the late effects associated with the administration of radiation alone or MOPP alone as a substantial fraction of patients achieved complete response and remained in their initial remission. Among all patients treated with radiation, 11 died in first remission (5 from a second cancer, 2 from CHF, 1 MI, 1 pulmonary fibrosis, 1 AIDS, 1 suicide). Among all patients treated with MOPP, 3 died in first remission (2 from a second cancer, 1 MI). The difference in survival after achieving initial CR (93% for MOPP, 78% for radiation) is significant (p=0.054). The current standard practice of using combined modality therapy might be expected to incur the toxicities associated with both chemotherapy and radiation therapy. Furthermore, synergistic toxicities associated with the combination have been described. Thus, efforts should be made to define the substantial fraction of patients whose disease can be controlled with a single modality and reserve combined modality therapy for the subset that requires it for disease control.
HD12 Randomised Trial Comparing 8 Dose-Escalated Cycles of BEACOPP with 4 Escalated and 4 Baseline Cycles in Patients with Advanced Stage Hodgkin Lymphoma (HL): An Analysis of the German Hodgkin Lymphoma Study Group (GHSG), University of Cologne, D-50924 Cologne, Germany.
Authors: Engert Andreas , Franklin Jeremy , Mueller Rolf-Peter , Eich Hans-Theodor , Gossmann Axel , Mueller-Hermelink Hans Konrad , Cerny Thomas , Markova Jana , Ho Anthony D. , Kanz Lothar , Greil Richard , Hiddemann Wolfgang , Pfistner Beate , Diehl Volker
Published: April 2020

The preceding trial HD9 had shown a significant superiority for escalated BEACOPP (EB) for failure-free survival (FFTF) and overall survival (OS) over COPP/ABVD or baseline BEACOPP (BB) (each 8 cycles). HD12 aimed to de-escalate chemotherapy by comparing 8 cycles EB with 4 cycles EB plus 4 cycles BB, with or without consolidatory 30 Gy radiation (RT) to initial bulky and residual disease. A total of 1661 patients aged 16–65 with HL in stage IIB (large mediastinal mass and/or E-lesions) or stage III-IV were randomised from 9/1999-1/2003 according to a factorial design between: 8EB + RT, 8EB no RT, 4EB+4BB + RT, 4EB+4BB no RT. A central diagnostic panel reviewed CT images after completion of chemotherapy independent of randomisation arm, and prescribed RT in selected cases. In the fifth analysis of HD12 (5/2006), 94% of all 1593 eligible randomised patients were evaluable, including 1498 for the chemotherapy comparison and 1449 for the radiotherapy comparison. Patient characteristics were very similar for all 4 arms. Radiation was given in arms with planned RT in 65%, in arms with no planned RT in 10%. 93% of all patients reached a complete remission, 2.2% suffered early progression, 4.6% relapsed and 8.2% died with a median folllow-up time of 48 months. Death due to acute toxicity was 3.2% (sepsis, cardiac, pulmonary, infection), with 21 such deaths in the 8EB arms and 27 in the 4EB+4BB arms. Secondary neoplasias were observed in 55 patients (3.7%): 14 AML/MDS, 12 NHL and 29 solid tumors/others. Four-year Kaplan-Meier estimates for arm pairs according to the factorial design were:

4 year rates, %(95% CI) 8EB vs. 4EB+4BB with RT vs. without RT FFTF 88(86–91) 86(83–89) 91(89–93) 88(86–91) OS 93(91–95) 91(89–93)

No significant differences were observed when comparing treatments according to the sequential analysis plan (N.B. 10% received RT in arms ‘without RT’). The final analysis, scheduled when 80% of patients have 5 years follow-up, must be awaited before final conclusions can be drawn.
Cancer Risks in Fanconi Anemia: Experience of the German Fanconi Anemia (GEFA) Registry.
Authors: Rosenberg Philip S. , Alter Blanche P. , Ebell Wolfram
Published: April 2020

OBJECTIVE: Acute Myeloid Leukemia (AML) and solid tumors (ST) occur frequently in Fanconi Anemia (FA). Our objective was to characterize the age of onset of cancer and identify any neoplasms occurring in excess.

METHODS: We analyzed data from the German Fanconi Anemia (GEFA) Registry, a retrospective cohort. For competing adverse events of bone marrow failure (BMF), AML, and ST, we determined cause-specific hazards and cumulative incidence curves. We calculated the ratio of observed to expected cancers (O/E ratio) in GEFA compared to age- and sex-matched persons from the general North American population. We studied outcomes after bone marrow transplantation (BMT) using survival analysis.

RESULTS: In GEFA, 182 patients contributed 2548 person-years of observation prior to BMT; 63 had BMF as the first adverse event, 15 had AML, and 10 had ST. The cumulative incidence by age 50 was 48% for BMF and 29% for ST. The cumulative incidence by age 20 was 9% for AML. The hazard of BMF peaked at 4%/y at age 10. The hazard of AML increased to 1.6%/y at age 20. The hazard of ST increased from 1%/y at age 20, to 5%/y at age 40, to ~10%/y at age 50. The O/E ratio was 45 for all cancers, 24 for all solid tumors, and 926 for AML; these increased risks were statistically significant. Significantly elevated O/E ratios were observed for esophagus (6346), vulva (2436), oral cavity and pharynx (121), breast (34), and brain (23) cancers. Forty-eight patients had BMT prior to cancer. Subsequently, there were 20 deaths and 3 malignancies in 216 person-years. The 3 malignancies (tongue, liver, and esophagus) occurred 2, 16, and 17 years after mismatched, matched, and matched transplant at ages 13, 23, and 34, respectively. The age-specific hazard of ST was 3.8-fold higher in transplanted versus untransplanted patients; this increased risk was not significant (P = 0.11). During 2000–2004, none of 5 patients with matched, and 3 of 18 patients with mismatched donors, died during the period from 0 – 6 months. In patients with matched donors, acute and chronic GVH were significant risk factors for death beyond 6 months.

CONCLUSIONS: Absolute and relative risks of cancer in GEFA are quantitatively similar to previously reported estimates from the North American Survey. Outcomes after transplantation in GEFA are comparable to the Hôpital St Louis Cohort. Our prior observation that FA patients who survive BMF are at extraordinary risk of specific ST has been replicated.
OFD1-Mediated T Cell Responses in MGUS Patients: Implications for Immunotherapy.
Authors: Blotta Simona , Prabhala Rao , Pelluru Dheeraj , Tetsuro Sasada , Franco Brunella , Tagliaferri Pierosandro , Tassone Pierfranceso , Anderson Kenneth C. , Munshi Nikhil C.
Published: April 2020

We have identified a panel of 11 novel antigens with induced antibody response in patients with Monoclonal Gammopathy of Undetermined Significance (MGUS) by screening a Multiple Myeloma (MM) cDNA library with patient serum using Serological Analysis of Recombinant Expression cloning (SEREX). Among these antigens, a specific antibody response against OFD1 protein was observed in 6/29 (20.6%) MGUS patients, while 0/12 newly diagnosed, 1/11 (8.3%) relapsed and 3/11 (27.2%) post-transplant MM patients in complete or partial remission showed an antibody response against this protein. Interestingly, in 1 of these patients the antibody response appeared only after autologous transplantation. No responses were observed in 25 healthy donors. OFD1 is widely expressed in embryonic stage with important role in organogenesis; however its expression decreases significantly in adult tissues. Moreover, OFD1 that is involved in the functional regulation of the Hedgehog-pathway, is dysregulated in solid tumors, suggesting a potential role in the tumorigenesis. We have observed the OFD1 expression at high levels in all 11 MM cell lines and 4 primary MM patient cells tested as compared with low or no expression in PBMC and normal bone marrow stromal cells. Specific sequences, derived from three clones corresponding to three different parts of OFD1 protein, were used to identify 15 peptides with potential binding to HLA-A*0201 and selected two peptides (3 and 8) on the basis of their high binding affinity. We observed presence of OFD1 specific CD8+ T cells, as measured by HLA-A2+ pentamers, on all 6 HLA2-A2+ MGUS patients tested. Presence of these cells was also observed in 5 MM patients. However, T cell proliferation following incubation with OFD1 peptides for 7days was observed only in MGUS (=6) while T cell proliferation was not observed in MM (n=5) patients indicating intact OFD1-specific responses in MGUS as compared with MM. These results highlight a possible role of OFD1 in the immune regulation in MGUS and provide the framework for further evaluation of OFD1 as a new potential target for immunotherapeutic approaches in both MGUS and MM.
Activation of the JNK Pathway Mediates Chemoresistance in T-Cell Acute Lymphoblastic Leukemia by Phosphorylation and Proteosomal Degradation of BimEL.
Authors: Leung Kam Tong , Li Karen Kwai Har , Sun Samuel Sai Ming , Chan Paul Kay Sheung , Wong Yum Shing , Ooi Vincent Eng Choon , Chiu Lawrence Chi Ming
Published: April 2020

Despite progress in the development of effective treatments against T-cell acute lymphoblastic leukemia (T-ALL), about 20% of patients still exhibit poor response to the current chemotherapeutic regimens and the cause of treatment failure in these patients remains largely unknown. In this study, we aimed at finding mechanisms that drive T-ALL cells resistant to chemotherapeutic agents. By screening etoposide sensitivity of a panel of T-ALL cell lines using DNA content and PARP cleavage as apoptosis markers, we identified an apoptosis-resistant cell line, Sup-T1. Western blot analysis and caspase activity assay showed that Sup-T1 cells were deficient in etoposide-induced activation of caspase-3 and caspase-9. In addition, mitochondrial cytochrome c release was not evident in etoposide-treated Sup-T1 cells. However, addition of exogenous cytochrome c in cell-free apoptosis reactions induced prominent caspase-3 activation, indicating that the chemoresistance observed in Sup-T1 cells was due to its insusceptibility to the drug-induced mitochondrial alterations. Analysis of the basal expression of the Bcl-2 family proteins revealed that the levels of Bcl-2 was higher in Sup-T1 cells, while Bax and BimEL levels were lower, when compared to etoposide-sensitive T-ALL cell lines. Gene silencing using antisense oligonucleotide to Bcl-2 and overexpression of Bax did not resensitize cells to etoposide-induced apoptosis. On the contrary, transient transfection of BimEL into Sup-T1 cells significantly restored etoposide sensitivity. Further experiments revealed that the lack of BimEL expression in Sup-T1 cells was due to the rapid degradation of newly-synthesized BimEL by the proteosomal pathway, as treatment of Sup-T1 cells with a proteosome inhibitor significantly restored the protein level of BimEL. Moreover, treatment with proteosome inhibitor resulted in mobility shift of BimEL, which was sensitive to phosphatase digestion. Furthermore, treatment of Sup-T1 cells with JNK inhibitor resulted in accumulation of BimEL, and pretreatment with JNK inhibitor restored sensitivity of Sup-T1 cells to etoposide-induced apoptosis, indicating that constitutive activation of the JNK pathway in Sup-T1 cells was responsible for promoting BimEL phosphorylation, and this may serve as a signal targeting BimEL to the proteosome for degradation. Altogether, our findings provide the first evidence that JNK activation correlates inversely with BimEL level by promoting its phosphorylation and degradation. This, in turn, reduces the sensitivity of T-ALL cells to chemotherapeutic agents.
Chromosome 1p21 Deletion Defines a Subgroup of Multiple Myeloma with Poor Clinical Outcomes Independent of del (13q), del(p53), t(4;14) or 1q21 Amplification.
Authors: Chang Hong , Qi Connie , Jiang Allan , Xu Wei , Young Trieu , Mikhael Joe , Trudel Suzanne , Chen Christine , Reece Donna
Published: April 2020

Amplifications involving chromosome 1q and deletions involving 1p are frequent events in multiple myeloma (MM). The pathogenesis and clinical significance of these anomalies is largely unknown but CKS1B amplification at 1q21 detected in 30–40% of MM patients is associated with disease progression. As karyotyping and SNP based mapping analysis identify a minimal common deletion region involving the 1p21 locus, we used FISH combined with cytoplasmic light chain detection (cIg-FISH) to investigate the prevalence and prognostic significance of del(1p21) in a cohort of 186 MM patients undergoing autologous stem cell transplant. CIg-FISH detected hemizygous 1p21 deletions in 18% of the cases. The median percentage of clonal plasma cells harboring del(1p21) was 55% (range 20–95%). The presence of 1p21 deletions was strongly correlated with CKS1B amplification (p=0.004), t(4;14) (p= 0.027), and del(p53) (p=0.04), but not with del(13q) or t(11;14). There was no association between del (1p21) and other biological factors including age, gender, Hb, albumin, C-reactive protein, beta-2 microglobulin level, isotype or bone marrow plasmacytosis. Patients with 1p21 deletions had significantly shorter progression-free (median 10.5 vs. 25.4 months, p=0.0001) and overall survivals (median 33.9 months vs. not reached, p=0.001) than those without such deletions. On multivariate analysis, del(1p21) was an independent risk factor for progression free (p< 0.0001) and overall survivals (p=0.0005) after adjusting for other genetic risk factors including del(13q), del(p53), t(4;14) and CKS1B amplification. Our results indicate that del(1p21) is a novel genetic risk factor and warrant inclusion of this genetic aberration in the risk-stratification of MM. Further studies are required to identify candidate tumor suppressor gene(s) at the 1p21 locus and explore their role in the molecular pathogenesis of MM.
The Clonal Inventory of Gene Corrected Hematopoiesis in Three Successful Clinical Gene Therapy Trials
Authors: Schwarzwaelder Kerstin , Schmidt Manfred , Deichmann Annette , Howe Steven J. , Ott Marion G. , Stein Stefan , Siler Ulrich , Hacein-Bey-Abina Salima , Glimm Hanno , Hoelzer Dieter , Cavazzana-Calvo Marina , Thrasher Adrian J. , Seger Reinhard , Grez Manuel , von Kalle Christof
Published: April 2020

To enhance the safety and efficacy of future gene therapy trials using integrating vector systems it is necessary to analyse the clonality of the genetically modified cell pool. The comparative analyses of integration site distribution and cellular gene expression will further reveal causal mechanisms of in vivo clone selection. We followed the repopulation clonality of 21 patients which participated in 3 successful clinical gene therapy trials via linear amplification mediated PCR (LAM PCR). We identified the integration sites (IS) of pre and post transplantation samples by Sanger sequencing and accomplished RNA analyses. The comparative results from all trials showed that vector integration is favoured in gene coding regions, in particular transcriptional start sites. In both X-SCID trials significantly more post transplantation IS were located in or in the vicinity of genes encoding proteins with kinase or transferase activity. In pre transplantation samples no uniform gene class was overrepresented. In both trials we detected common insertion sites mainly post transplantation and the effect was more pronounced in the trial where 4 patients developed vector induced leukemia. Notably, we detected no significant differences regarding the IS distribution in leukemic versus non leukemic patients. The gene corrected repopulation of patient 1 and 2 of the X-CGD trial was polyclonal until 542 and 777 days after transplantation, respectively. 5 months after therapy dominant clones appeared. In patient 1, between 616 and 820 days post transplantation (post mortem time point) the number of participating clones and the contribution of a dominant clone decreased while the contribution of another dominant clone increased. In both patients the integrated vector induced the upregulation of the genes MDS1/EVI1, PRDM16 or SETBP1 and thus led to the in vivo expansion of affected cell clones. From these trials we sequenced >2000 unique IS by Sanger sequencing and several thousand via pyrosequencing (datamining is ongoing). Our data show that the integration site distribution was non random, that the integrated vector influenced the cellular gene expression which caused subtle to massive changes in the repopulation clonality and that it will soon be possible to define the clonal inventory of patients using next generation sequencing technologies.
Deferasirox Might Induce Immunosuppression Comparable to Deferoxamine.
Authors: Jeong Dae-Chul , Chung Nack-Gyun , Cho Bin , Kim Sung-Yong , Han Chi Wha , Kim Hack Ki
Published: April 2020

Background: The iron chelating agents (ICA) have various biological effects besides iron chelation, including induction of apoptosis and immune modulation. Deferoxamine (DFO) is known to have some immunosuppressive effects. Deferasirox (DFS) is a new oral iron chelating agent, and its biologic effects are not known clearly. We investigated the immunosuppressive effects of DFS in comparison with DFO.

Materials and Methods: We obtained spleen cells (SP) from 5 week-old female C57BL/6 (B6, H-2b) and BLAB/c (H-2d). The cytotoxicity of ICA at various concentrations was examined by CCK8 method for 48 hours. The cell proliferation assay (CPA) was done with B6 SP and irradiated BALB/c SP under various concentrations of DFO or DFS with phytohemaglutinin by ELISA method (n = 6). Stimulation index (SI) was defined as the ratio of experimental OD compared with OD of control. Experimental design was as follows: control (without any treatment), DFO 5, 10, 50, 100 μM, and DFS 15, 30, 50, 100 μM. Also, CPA was performed after 12 hours incubation with 20 μM ferric chloride (FE) (n = 6). For cytokine analysis, IL-2, IL-10, and TGF-β were measured from supernatants collected during CPA.

Results: There was over 100% viability after 48 hours of incubation at various concentrations, and similarly under FE. In DFO group, there was inhibition of cell proliferation at all concentrations with or without FE. Inhibition was greater at DFO10 and DFO50 than others with treatment of FE (P < 0.01). In DFS group, SI without FE diminished regardless of concentration. SI with FE showed significant decrease at DFS levels higher than 30mM (P < 0.01). Decrement of SI in DFS group was comparable to that in DFO group. TGF-b and IL-10 levels were not significant at any concentration of both ICAs (P > 0.05). However, IL-10 was diminished according to DFS or DFO concentration without statistical significance (P > 0.05). In DFS group, the level of IL-2 was higher at 30 μM than others (P < 0.01). In DFO group, IL-2 level did not change according to concentration.

Conclusion: These data suggest that ICAs may have immune suppressive effects which may not be dependent on cytokines. We conclude that DFS, a new oral iron chelating agent, induce immunosuppression comparable to DFO.
Evaluation of Deferasirox (Exjade®, ICL670) Therapy in Patients with Transfusional Iron Overload Who Achieve Serum Ferritin (SF) ≤1000 ng/mL in Long-Term Studies.
Authors: Cappellini Maria Domenica , Vichinsky Elliott , Ford John M. , Rabault Bertrand , Porter John
Published: April 2020

Background: Maintaining SF levels below 1000 ng/mL offers significant clinical benefits in patients with transfusional iron overload. However, low SF levels can be associated with an increased risk for iron chelator toxicity. This analysis was performed to assess safety parameters during deferasirox treatment in patients with transfusion-dependent anemias who reached SF levels ≤1000 ng/mL on at least two consecutive visits during the extension phases of four core studies.

Methods: Patients (≥2 years old) with β-thalassemia, MDS, sickle cell disease or other transfusion-dependent anemias enrolled in four clinical trials were treated for 1 year with deferasirox 5–30 mg/kg/d, followed by extension phases of 4 years. In the extension phases, patients either continued receiving deferasirox (deferasirox cohort) or crossed over from DFO to deferasirox (crossover cohort), while receiving dose modifications based on efficacy and safety parameters. Efficacy was monitored by monthly SF readings; safety was assessed by the incidence and type of adverse events (AEs).

Results: Following dose adjustments at the end of the core trials, 163/652 (25%) patients in the deferasirox cohort reached SF levels ≤1000 ng/mL on at least two consecutive visits after a median of 1.2 years on deferasirox therapy. Median SF levels over the course of the studies are presented in Table. At month 22, the overall mean dose was 20 mg/kg/d, which was generally maintained in all dose groups until month 42. At baseline, overall mean (±SD) ALT and AST values were 44.2 (36.2) and 39.2 (27.4) U/L, respectively. Overall ALT and AST levels decreased by 16.5 (±38.7) and 11.7 (±30.5) U/L, respectively, at month 42.

Median SF (ng/mL) Initial dose group Month 5 and 10 mg/kg/d 20 mg/kg/d 30 mg/kg/d (n) (n) (n) *Dose modifications to most appropriate dose Baseline 1295 (55) 1520 (43) 2311 (65) 1 1475 (54) 1616 (42) 2284 (65) 6 1612 (54) 1383 (39) 1596 (60) 12* 1697 (53) 1165 (42) 1168 (62) 18 1818 (45) 1362 (39) 1307 (51) 24 1287 (49) 1161 (34) 1236 (52) 30 966 (44) 1046 (33) 1047 (54) 36 788 (37) 1010 (30) 991 (50) 42 884 (37) 1040 (34) 850 (50)

10 (6.1%) patients experienced two consecutive serum creatinine increases >33% above baseline and >ULN. Most of these increases were only marginally >ULN and none were >2 x ULN. Creatinine increases were non-progressive and generally fell promptly following dose reductions of 5–10 mg/kg/d. No new AEs or safety concerns have been reported thus far in this population.

Conclusions: With appropriate deferasirox dosing, iron overloaded patients can effectively achieve and maintain low SF levels. There were no major safety issues observed in achieving SF levels of ≤1000 ng/mL in this population. Decreases in liver enzymes suggest improvement in liver function, probably as a result of reduced iron burden.
Building and Clinical Use of Activated Plasma Clotting Time Test and the Value of APCT on Predicting the Bleeding Due to Leukemia Chemotherapy-Induced Thrombocytopenia.
Authors: Chen Bao-An , Li Jin-Jin , Huang Cheng-Yin , Gao Feng , Chen Jian , Gao Chong , Sun Yun-Yu , Wang Jun , Zhao Gang , Ding Jia-Hua , Chen Ning-Na , Pei Xiao-Ping
Published: April 2020

Objective This study was aimed to determine the optimal concertration of C-PG in activated plasma clotting time (APCT) test and build the APCT test.To investigate the value of activated plasma clotting time test (APCT) on predicting the bleeding due to Leukemia chemotherapy-induced thrombocytopenia.

Method We collected healthy blood donors’venous blood, prepared watched platelet suspension and platelet rich plasma in a normal way to which we add different concertrations of C-PG, then test expression of AnnexinV in the surface of activated platelet by FCM. Prepared platelet rich plasma and platelet poor plasma in a normal way to which we add different concertrations of C-PG, then count the plasma clotting time.Forty-one patients with leukemia chemotherapy, twenty-one patients with petechiae or epistaxis or gum bleeding and twenty patients without bleeding symptoms, were involved in the test. Drawed their venous blood and collected in 3.2% buffered sodium citrate(9:1), the platelet counts and APCT, PT, APTT, TT, Fig were assesed.

Result Bleeding group’s APCT was obviously prolonged contrast to the non-bleeding group’s. Platelet counts and APCT of the bleeding group and the non-bleeding group were (7.2±2.9) ×109/L, (105.9±8.1)s and (23.0±12.2) ×109/L,(71.4±9.0)s respectively. There was significant difference between the two groups (P<0.01). Using APCT≥90s as the cut-off point to predict the bleeding caused by chemotherapy-induced thrombocytopenia, the sensibility was 100%, and the specificity was 95.7%. Using platelet counts ≤15*109/L, the sensibility and the specificity was 95.2% and 69.0%. PT, APTT, TT, Fig of the bleeding group and the non-bleeding group were 11.7±1.1)S,(31.5±1.3)S,(11.1±1.2)S,(2.37±0.41)g/L and (12.1±0.8)S,(30.8±2.1)S,(10.7±0.9)S,(2.64±0.27)g/L respectively. The results of the two groups were all not significantly different (P>0.05).

Conclusion APCT is a good indication of predicting the bleeding due to leukemia chemotherapy -induced thrombocytopenia.
Reduced Bone Mineral Density in Hemophilia.
Authors: Alrustamani Ahmad , Walker Irwin
Published: April 2020

Rationale: Hemophilias are hereditary bleeding disorders usually presenting with muscloskeletal bleeding (for example, hemarthroses, synovitis and hematomas). Recently it has been suggested that patients with hemophilia are at increased risk of osteoporosis. The arthropathy associated with hemophilia, along with the treatments for human immune defeciency virus (HIV) and hepatitis C virus (HCV) may predispose affected individuals to osteoporosis and pathologic fractures.

Methods: This was a retrospective study that included unselected male patients aged 18 yrs old and over with severe hemophilia managed and followed in the Hamilton-Niagara Regional Hemophilia Program. All patients have been recommended to have bone mineral density studies (BMD). BMD was performed using dual x ray absorptiometry.

Results: There were 19 patients, mean age 29 years, range 18–43. Prophylactic treatment with recombinant factors was received by 12/19 patients (63%); HCV infection was present in 14/19 (73%) and HIV in 6/19 (31%) patients, the latter all receiving HAART treatment. A reduced BMD (osteopenia) as defined by the World Health Organization was detected in 42% of patients (5/13 or 38% of Hemophilia A and 3/6 or 50% of Hemophilia B participants). Among those who received prophylactic recombinant factors, 5/12 had reduced BMD. There was a trend towards decreased BMD in patients with HIV or HCV.

Conclusions: Individuals with severe hemophilia are at increased risk of having low bone mineral density. Those with coexistent HIV and HCV infections may be at higher risk. There was no clear correlation in this small series between BMD and age, and the use of prophylactic factor concentrate. Further study is required to obtain a more precise prevalence of disease, and clarification of the risk factors; however, a high prevalence of osteopenia in such a young cohort of individuals is concerning and estimation of BMD in all individuals with severe hemophilia is advised.
Final Results of a Phase II Danish Trial Testing Low Dose Total Body Irradiation Following Six Bi-Weekly RCHOP-14 Plus 2 Extra Rituximab in Elderly High risk Patients with Aggressive CD20+ Diffuse Large B-Cell Lymphoma (DLBCL).
Authors: Safwat Akmal , Specht Lena , Hansen Flemming , Hansen Mads , Jurlander Jesper , d’Amore Francesco
Published: April 2020

Background: To further improve the results achieved by adding Rituximab (R) and shortening chemotherapy interval in elderly patients, an effective but relatively non-toxic treatment modality is needed. We tested therefore the addition of low dose total body irradiation (LTBI) of 1,6 Gy given after chemo-immunotherapy.

Methods: A multicenter, phase II trial including patients >60 yrs with stage II-IV, CD20-positive DLBCL was performed between 2003 and 2007. Patients received 6x R-CHOP-14 + 2x R alone followed by LTBI given as 2 courses of 4 daily fractions of 0,2 Gy separated by 2 weeks of rest. Radiotherapy to sites of bulky (>7.5 cm) disease was given according to the local guidelines of the participating centres.

Results: Forty two patients were included. Observation time ranged from 3 to 47 months with median follow up of 24 months. The median age was 67 years; 62% had stage III or IV; 48% had B symptoms and 36% had bulky (> 7.5 cm) disease; 50% had ECOG score ≥ 1; 76% had elevated LDH and 57% had IPI of >2. Twenty four patients (57%) achieved a CR or CRu at the end of chemotherapy, while 12 (28.5%) were in PR. One of the 12 PR patients refused LTBI. Of the remaining 11 PR patients who received LTBI, 8 (82%) achieved CR in the first follow up after LTBI while the remaining 3 patients had initially stable disease but progressed shortly after. One patient (2%) progressed under chemotherapy while seven patients (17%) relapsed after achieving CR. Six of these refractory/relapsed cases presented with IPI ≥ 3. The 3-yr event-free and progression-free survival values were 64.8% (SE: 8.7%) and 73.5% (SE: 8.9%), respectively, while the 3-yr overall survival was 85.4% (SE: 5.5%). There were 3 toxic deaths (7.1%) due to sepsis occurring during chemotherapy. Ninteen of 235 cycles of CHOP (8%) were given at reduced dose levels in 3 patients (7%), while 3 cycles (1.3%) were delayed but given at 100% dose level. None of the 305 injections of Rituximab were dose reduced. Only one of 31 patients (3%) got his second LTBI cycle at 75% of the planned dose because of thrombocytopenia. CTC Gr 3–4 neutropenia occurred following 50 of 250 R-CHOP-14 cycles (20%). CTC Gr. 3–4 thrombocytopenia was seen in 8 pts (22%) following LTBI.

Conclusions: Despite the high risk profile of the patient cohort enrolled in this trial, the 3-yr outcome values match the results of the best performing recent phase III clinical trials designed for elderly patients with DLBCL. Adding LTBI to 6 cycles of R-CHOP-14 was well tolerated and effective in converting the majority of PRs into CRs. Therefore, it may provide survival benefit and should be tested in a randomised setting.
The Pattern of KIR Ligands in Adult Patients with AML Is Associated with Various Post-Transplant Complications, Including GvHD after HLA-Matched Sibling or Unrelated Donor Transplantation
Authors: Kim Heeje , Min Woo-Sung , Cho Byung-Sik , Kim Yoo-Jin , Lee Seok , Min Chang-Ki , Cho Seok-Goo , Lee Jung-Lim , Lee Jong-Wook , Kim Chun-Choo
Published: April 2020

The function of natural killer (NK) cells in allogeneic stem cell transplantation (allo-SCT) is known. HLA-Cw is predominantly involved in inhibiting and activating human NK cells. Many pilot studies have examined the treatment of high-risk AML patients in terms of killer cell immunoglobulin-like receptor (KIR) disparity. NK alloreactivity may be determined by KIR and their ligands. We evaluated 370 adult AML patients scheduled for allo-SCT from family or unrelated donors. All but 41 patients in this study were in first complete remission (CR), as assessed just before the elective SCT. The HLA data for each transplant pair and clinical outcome were analyzed to study the impact of KIR ligands, especially on graft-versus-host disease (GvHD). High-resolution HLA typing was performed in 185 cases and serological data were used for the others. SCT was from an unrelated donor in 113 patients. Interestingly, we found a predominant pattern of group 1 (C1 epitope) HLA-Cw antigen in Korean acute myelogenous leukemia (AML). Only 3% of the pairs studied were C2 homozygotes. Heterozygosity of HLA-Cw C1 or C2 in either donors or recipients was closely related to the development of acute GvHD (P=0.006) and a lower rate of relapse (P=0.015). As in previous studies, we found discrepant results between the ligand-ligand model and the receptor-ligand model in the context of GvHD directed by allogeneic NK cells. We suggest that KIR ligands in allo-SCT, even in HLA-matched siblings or unrelated donors, are unique and a stable parameter to be considered in association with various post-transplant complications. According to the HLA immunogenetic background, KIR ligands are responsible for GvHD. Further investigations with longer follow-up are needed to verify the role of KIR receptor-ligand balance.