bluuebunny/crossref_metadata_2025 huggingface dataset:
WP-1066, a Next-Generation Member of JAK-Stat Inhibitors, Induces Cell Cycle Arrest, Abrogates Proliferation, and Induces Apoptosis of Acute Myeloid Leukemia (AML) Cells.
Authors: Ferrajoli Alessandra , Faderl Stefan , Van Quin , Harris David M. , Priebe Waldemar , Kantarjian Hagop M. , Talpaz Moshe , Estrov Zeev
Published: April 2020

Janus kinases (JAK) are tyrosine kinases associated with both cytokine receptors and downstream signal transducer and activator of transcription (Stat) proteins. Upon activation of JAK by a variety of cytokines and growth factors, Stats translocate to the nucleus and promote transcription of target genes. Constitutive activation of Stat proteins in AML has been associated with poor prognosis and AG490, an inhibitor of this pathway, was shown to suppress AML cell proliferation in vitro. WP-1066 represents a further development of AG490 with biological activity at significantly lower concentrations. Therefore, we studied the effects of WP-1066 on the AML cell lines OCIM2 and K562 and on fresh bone marrow aspirates obtained from five newly diagnosed AML patients. We found that WP-1066 inhibited the proliferation of OCIM2 and K562 cells and of fresh marrow AML blast colony-forming cells in a dose-dependent fashion at concentrations ranging from 0.5 to 3 μM. WP-1066 completely abrogated the growth of leukemia cells at a concentration of 3 μM. Furthermore, WP-1066 induced a cell cycle arrest of OCIM2 and K562 cells. Incubation of AML cells with 2 μM of WP-1066 resulted in a time-dependent accumulation of OCIM2 and K562 cells in the sub-G0 phase of the cell cycle. Those leukemia cells underwent apoptotic cell death as assessed by annexin V-FITC. Incubation of OCIM2 cells with 0.5 to 3 μM WP-1066 for 2 hours induced a dose-dependent apoptosis in 52% of the cells. A 4 hour exposure of either OCIM2 or K562 cells to 2 μM of WP-1066 induced caspase 3 activation and PARP cleavage. As expected, WP-1066 inhibited Stat3 and Stat5 phosphorylation in K562 and OCIM2 cells both in a time- and dose-dependent manner, confirming that inhibition of the JAK-Stat pathway is its mechanism of action. Overall, our data showing that WP-1066 inhibits the JAK-Stat pathway, suppresses proliferation, induces cell cycle arrest and apoptosis of AML cells, suggest that the activity of this compound warrants further exploitation aimed at developing WP-1066 for future therapy of AML.
Cyclosporine A Modulates the Multidrug Resistance Proteins P-Glycoprotein, Multidrug Resistance Protein, Breast Cancer Resistance Protein and Lung Resistance Protein.
Authors: Qadir Misbah , O’Loughlin Kieran L. , Williamson Nicole A. , Minderman Hans , Baer Maria R.
Published: April 2020

Multidrug resistance (MDR) mediated by the ATP-binding cassette proteins P-glycoprotein (Pgp), multidrug resistance protein (MRP-1), breast cancer resistance protein (BCRP) and the vault protein lung resistance protein (LRP) is implicated in treatment failure in acute myeloid leukemia (AML). Pgp, MRP-1 and BCRP mediate energy-dependent cellular drug efflux, while LRP blocks cytoplasmic-nuclear drug transport. MDR modulators are non-cytotoxic drugs that block the activity of MDR proteins. In clinical trials, the Pgp modulator cyclosporine A (CsA) improved treatment outcome in AML, while its non-immunosuppressive, non-nephrotoxic analogue PSC-833 has not, despite being a potent Pgp modulator in preclinical models. CsA is known to also modulate MRP-1, and we hypothesized that a broad spectrum of MDR modulation might contribute to its clinical efficacy. We studied the effects of CsA and PSC-833 on in vitro drug uptake, retention and cytotoxicity in drug-selected and transfected resistant cell lines overexpressing Pgp, MRP-1 or BCRP, and on nuclear-cytoplasmic drug distribution and cytotoxicity in cell lines overexpressing LRP. Cellular drug content was measured by flow cytometry, and nuclear-cytoplasmic drug distribution was assessed by confocal microscopy. CsA enhanced uptake and retention of the substrate drug mitoxantrone in cells overexpressing Pgp (HL60/VCR), MRP-1 (HL60/ADR) and BCRP (8226/MR20) and increased cytotoxicity 7-, 4- and 4-fold, respectively. Moreover, CsA increased the nuclear content of doxorubicin in 8226/MR20 cells, which co-express LRP with BCRP, and increased doxorubicin cytotoxicity 12-fold. The effect of CsA on nuclear drug content and cytotoxicity in 8226/MR20 cells occurred without an effect on cellular doxorubicin content, consistent with the fact that 8226/MR20 cells co-express wild type BCRP (BCRPR482), which does not efflux doxorubicin. Moreover the BCRP modulator fumitremorgin C had no effect on 8226/MR20 content, nuclear uptake nor cytotoxicity of doxorubicin. CsA also enhanced nuclear doxorubicin content in a second cell line with LRP-mediated resistance, HT1080/DR4. PSC-833 enhanced mitoxantrone retention and cytotoxicity in cells overexpressing Pgp, but, in contrast to CsA, had no effect on mitoxantrone uptake, retention nor cytotoxicity in cells expressing MRP-1 nor BCRP, and had no effect on doxorubicin nuclear content nor cytotoxicity in cells expressing LRP. Thus CsA is a broad-spectrum MDR modulator, with activity against Pgp, MRP-1, BCRP and LRP, while PSC-833 only modulates Pgp. The broad-spectrum activity of CsA may contribute to its clinical efficacy. These findings support identification and testing of other broad-spectrum MDR modulators.
Murine Mesenchymal Stem Cells Improve Haemopoietic Engraftment in a Murine Transplant Model and Is Maximised through Osteogenic Stimulation.
Authors: Grainger John D. , Fairbairn Lez J. , Wynn Robert F.
Published: April 2020

Several studies have preciously shown improved engraftment of human haemopoietic stem cells (HSC) following co-infusion of human mesenchymal stem cells (MSC). However, these studies have been criticised for using xenogeneic recipients which might support human haemopoiesis through the production of species-specific cytokines and growth factors by human MSC. To further investigate the potential for MSC to support HSC engraftment we used a murine to murine transplant model to co-infuse purified murine MSC and murine HSC.

The MSC were derived from collagenase-treated bone fragments of the Rosa26 murine strain, had the phenotype CD45- CD31-CD11b- Sca-1bright VCAM1+ and could differentiate into osteocytes, chondrocytes and adipocytes. Donor and recipient HSC were obtained from unmanipulated bone marrow of either the C57Bl/6J murine strain, CD45 isotype CD45.2, or the PEP3b murine strain, expressing CD45.1. Following 10Gy irradiation to the recipients HSC and MSC were co-infused. Donor and recipient haemopoietic engraftment in the peripheral blood was quantified by FACS analysis of the CD45.1/CD45.2 ratio.

Successive transplant experiments persistently showed improved donor haemopoietic engraftment following co-infusion of 2 x 105 BM cells and 1 x 106 MSC (three seperate transplant series p=0.001, p=0.09 and p=0.13). The beneficial effect was maximal at the lower HSC dose of 1 x 105 BM cells (p=0.004, p=0.045 and uninterpretable in the third transplant series due to excessive death observed in the non-MSC recipients).

Increasing the MSC dose to 2 x 106 cells showed a non-significant increase in survival and donor haemopoietic engraftment but further MSC escalation resulted in fatal emboli post-infusion.

LacZ staining of tissue sections failed to show evidence of MSC outside the lungs but was detected at very low levels in the bone from one recipient.

As MSC are osteoblast precursors and recent literature suggests a role of osteoblasts as the HSC niche we investigated the effect of pre-culturing our MSC population in osteogenic media prior to transplant. Following 16 days of culture approximately 40% of colonies showed ALP activity. At this time point osteogenic-stimulated MSC (O-MSC) were removed from culture, washed to remove any residual osteogenic media, and infused in our standard method. O-MSC recipients receiving 1 x 105 BM cells showed a mean engraftment of 79% compared to 21% in non-msc recipients receiving 2 x 105 BM cells (p=0.000017), and 42% in recipients receiving standard MSC and 1 x 105 BM cells (p=0.01).

In conclusion we confirm improved haemopoietic engraftment in a non-xenogeneic model and does not require significant MSC engraftment. The beneficial effect is maximised through osteogenic stimulation of MSC suggesting the possibility that the mechanism may be through priming of the HSC niche.
Human Memory T Cells Proliferate but Do Not Elicit Cytotoxicity in Response to Alloantigens.
Authors: Chen Benny J. , Cui Xiuyu , Chao Nelson J.
Published: April 2020

We and others have recently demonstrated that memory T cells do not induce graft-versus-host disease in several different animal models. To test whether the same concept applies to humans, we compared the ability of memory T cells to respond to alloantigens with that of naive as well as bulk T cells. Purified T cells were first obtained from peripheral blood from healthy donors and then separated into memory and naive T cell subsets based on the expression of CD45RA (memory: CD45RA−, naive: CD45RA+). Memory T cells were subsequently tested for their ability to respond to alloantigens using proliferation and cytotoxicity assays in comparison with naive and bulk T cells. Proliferation assay was performed using 1.25x105 responder cells and 5x105 irradiated stimulator cells per well in 96-well flat-bottom plate. Cytotoxicity was measured by the standard 4-hour Cr-51 release assay after 5-day mixed lymphocyte culture. In contrast to the mouse data, memory T cells proliferated equally well as naive and bulk T cells did in mixed lymphocyte culture (Figure A). However, these same memory T cells failed to kill the allogeneic targets despite the vigorous proliferative responses against the same alloantigens (Figure B). These data demonstrated that human memory T cells proliferate but do not elicit cytotoxicity in response to alloantigens, suggesting that human memory T cells may not contain true alloantigen-specific T cells if they have never exposed to those alloantigens before and may not cause graft-versus-host disease upon in vivo transfer. These observations warrant the further testing of human memory T cells in clinically more relevant models and in vivo for their ability to induce graft-versus-host disease.

Figure Figure
Dominant T-Cell Clonal Expansion Associated with Acute Graft-Versus-Host Disease after Allogeneic Hematopoietic Stem Cell Transplantation in Pediatric Patients.
Authors: Chen Xiaohua , Knowles James , Barfield Raymond , Lawrence Dawn , Handgretinger Rupert
Published: April 2020

Allogeneic hematopoietic stem cell transplantation (AHSCT) provides a valuable therapy for a variety of malignancies, solid tumors, hematological disorders and autoimmune diseases. One of the major complications in recipients after transplantation is graft-versus-host disease (GvHD), which can cause delayed T-cell reconstitution and therefore increase the risk of infection and mortality. It has been suggested that T cells play an important role in the development of GvHD. Several previous studies have identified T-cell clones associated with acute GvHD in adult patients. However, T-cell clonal expansion in the early stage of post-transplantation might also be caused by T-cells which are unrelated to acute GvHD. In this study, we used TCRβ CDR3 size spectratyping to detect T-cell clonal expansion among the polyclonal complexities of peripheral blood. We examined the TCRβ repertoire distribution at the onset of acute GvHD in 10 pediatric patients. We then compared that result with the TCRβ pattern at the same post-AHSCT stage in another 10 pediatric patients without acute GvHD.

Method: An RT-PCR assay for Vβ CDR3 size distribution was performed. Each fluorescent PCR product was detected by an automated 310 DNA sequencer. The data were analyzed by GeneScan software.

Results and Discussion: As expected, the TCRβ repertoire distribution was much skewed for all patients in both groups, because the immune suppression occurred at early stage of post-AHSCT. There were about 4–8 T-cell clones expanded in each patient for both groups. The TCRβ subfamilies among those clones varied significantly from patient to patient. However, Vβ2, Vβ9, Vβ13 and Vβ20 were commonly found in both groups, indicating in these cases that clonal expansion was not related to the occurrence of acute GvHD. Notably, TCR Vβ16 was found in 9 of 18 samples from 7 of 10 patients with acute GvHD. This TCR subfamily was not dominant in the patients without acute GvHD. This finding strongly suggests that the T-cell clone carrying TCR Vβ16 subfamily might be associated with acute GvHD in pediatric patients. The characterization of this T-cell clone is under study.
Rb Regulates Erythroid Differentiation through Bcl-XL-Dependent Anti-Apoptotic Effect.
Authors: Matsuoka Sahoko , Hirao Atsushi , Arai Fumio , Ito Keisuke , Takubo Keiyo , Nomiyama Kana , Hamaguchi Isao , Ikeda Yasuo , Suda Toshio
Published: April 2020

Inactivation of the retinoblastoma (Rb) gene results in embryonic lethality due to severe anemia and increased nucleated erythrocytes by day E14.5. However, molecular mechanisms of the function of Rb in erythroid differentiation have been unclear. Recent studies have suggested that Rb has both intrinsic and extrinsic roles on erythroid differentiation. Here we showed that Rb regulates terminal erythroid differentiation through inhibition of apoptosis. Enucleation of erythroblasts was impaired in semisolid culture of Rb−/− hematopoietic progenitors in fetal liver. The lethally-irradiated recipient mice transplanted with Rb−/− hematopoietic stem cells (HSCs) showed severe anemia (Hgb 8.2±1.47 g/dl versus WT :12.0±1.22 g/dl) with splenomegaly, whereas the number of leukocytes and platelets were normal. In Rb−/− recipient mice, the nucleated erythrocytes and reticulocytes were significantly increased in the peripheral blood. We analyzed cell surface markers for erythroid lineage (TER119 and CD71) in the enlarged spleen. A block of erythroid differentiation at the early erythroblast stage, accompanied with increased apoptosis, was observed in the recipient mice with Rb−/− HSCs. We speculated that the defect in the erythroid differentiation of Rb−/− HSCs might be caused by inappropriate cell death. Thus, we examined expression profiling for apoptosis-related genes in early erythroblasts (CD71highTer119high) by RT-PCR. Interestingly, the dramatic decrease of Bcl-XL expression was detected in the erythroblasts, whereas other Bcl-2 family members including Bcl-2 and Bax were unchanged. To clarify the function of Bcl-XL, we introduced exogenous cDNA of mouse Bcl-XL with GFP (Bcl-XL ires GFP) or GFP alone as control into HSCs and then transplanted them to lethally irradiated mice. From the point of CD71 and Ter119 expression pattern in GFP positive cells, Rb−/− erythoblasts still showed the block in differentiation. In contrast, a ratio of late erythroblasts in Bcl-XL expressing cells was higher than that in GFP control cells in Rb−/− recipient mice and a number of annexin V positive cells were much reduced in the mice transplanted with Bcl-XL expressing cells, indicating that overexpression of Bcl-XL inhibited inappropriated apoptosis and restored the differentiation. These data demonstrates that Rb regulates erythroid differentiation through Bcl-XL-dependent anti-apoptotic effect.
A Role for NIPA in DNA Damage Response.
Authors: von Klitzing Christine , Bassermann Florian , Morris Stephan W. , Peschel Christian , Duyster Justus
Published: April 2020

The nuclear interaction partner of ALK (NIPA) is a nuclear protein identified by our group in a screen for NPM-ALK interaction partners. We recently reported that NIPA is an F-box protein that assembles with SKP1, Cul1 and Roc1 to establish a novel SCF-type E3 ubiquitin ligase. The formation of the SCFNIPA complex is regulated by cell cycle-dependent phosphorylation of NIPA that restricts SCFNIPA assembly from G1- to late S-phase, thus allowing its substrates to be active from late S-phase throughout mitosis.

Proteins involved in cell cycle regulation frequently play a role in DNA damage checkpoints. We therefore sought to determine whether NIPA has a function in the cellular response to genotoxic stress. For this reason we treated NIH/3T3 cells with various DNA-damaging agents. Surprisingly, we observed phosphorylation of NIPA in response to some of these agents, including UV radiation. This phosphorylation was cell cycle phase independent and thus independent of the physiological cell cycle dependent phosphorylation of NIPA. The relevant phosphorylation site is identical to the respective site in the course of cell cycle-dependent phosphorylation of NIPA. Thus, phosphorylation of NIPA upon genotoxic stress would inactivate the SCFNIPA complex in a cell cycle independent manner. Interestingly, this phosphorylation site lies within a consensus site of the Chk1/Chk2 checkpoint kinases. These kinases are central to DNA damage checkpoint signaling. Chk1 is activated by ATR in response to blocked replication forks as they occur after treatment with UV. We performed experiments using the ATM/ATR inhibitor caffeine and the Chk1 inhibitor SB218078 to investigate a potential role of Chk1 in NIPA phosphorylation. Indeed, we found both inhibitors to prevent UV-induced phosphorylation of NIPA. Current experiments applying Chk1 knock-out cells will unravel the role of Chk1 in NIPA phosphorylation. Additional experiments were performed to investigate a function for NIPA in DNA-damage induced apoptosis. In this regard, we observed overexpression of NIPA WT to induce apoptosis in response to UV, whereas no proapoptotic effect was seen with the phosphorylation deficient NIPA mutant. Therefore, the phosphorylated form of NIPA may be involved in apoptotic signaling pathways.

In summary, we present data suggesting a cell cycle independent function for NIPA. This activity is involved in DNA damage response and may be involved in regulating apoptosis upon genotoxic stress.
Interaction of Stem Cells and Their Niche: Behavior and Gene Expression Profiles of CD34+/CD38− Cells upon Co-Cultivation with AFT024.
Authors: Wagner Wolfgang , Saffrich Rainer , Wirkner Ute , Eckstein Volker , Blake Jonathon , Ansorge Alexandra , Schwager Christian , Miesala Katrin , Ansorge Wilhelm , Ho Anthony D.
Published: April 2020

Cell-cell contact between stem cells and cellular determinants of the microenvironment plays an essential role in the regulation of self-renewal and differentiation. The stromal cell line derived from murine fetal liver (AFT024) has been shown to support maintenance of primitive human hematopoietic progenitor cells (HPC) in vitro. We have studied the interaction between HPC (defined as CD34+/CD38− umbilical cord blood cells) and AFT024 and the impact of co-cultivation on the behavior and gene expression of HPC. By time lapse microscopy the mobility and behavior of CD34+/CD38− cells were monitored. Approximately 30% of the CD34+/CD38− cells adhered to the cellular niche through an uropod. CD44 and CD34 were co-localized at the site of contact. Gene expression profiles of CD34+/CD38− cells were then compared upon co-cultivation either with or without AFT024. After cultivation for 16h, 20h, 48h or 72h the HPC were separated form the feeder layer cells by a second FAC-Sort. Differential gene expression was analyzed using our Human Genome cDNA Microarray of over 51,145 ESTs. Among the genes with the highest up-regulation in contact with AFT024 were several genes involved in cell adhesion, proliferation and DNA-modification including tubulin genes, ezrin, complement component 1 q subcomponent 1 (C1QR1), proto-oncogene proteins c-fos and v-fos, proliferating cell nuclear antigen (PCNA), HLA-DR, gamma-glutamyl hydrolase (GGH), minichromosome maintenance deficient 6 (MCM6), uracil-DNA glycolase (UNG) and DNA-methyltransferase 1 (DNMT1). In contrast, genes that were down-regulated after contact with AFT024 included collagenase type iv (MMP2), elastin (ELN) and hemoglobin genes. Differential expression of six genes was confirmed by RT-PCR. Other authors have reported on the differential gene expression profiles of CD34+ cells derived from the bone marrow versus those from G-CSF mobilized blood. As CD34+ cells from the bone marrow might represent cells exposed to the natural HPC niche we have then compared our findings with these experiments. In these comparisons we identified several overlapping genes that are involved in regulation of cell cycle and DNA repair including PCNA, DNMT1, MCM6, MCM2, CDC28 protein kinase regulatory subunit 1B (CKS1B), Topoisomerase II (TOP2a), DNA Ligase 1 (LIG1) and DNA mismatch repair protein MLH1. All these genes were up-regulated among CD34+/CD38− cells upon co-culture with AFT024, as well as among CD34+ cells derived from the bone marrow versus those from peripheral blood. Our studies support the hypothesis that intimate contact and adhesive interaction of HPC with their niche profoundly influenced their proliferative potential and their differentiation program.
Tie-2 Activation Is Required for Regeneration of Marrow Vasculature, Supporting Hematopoietic Reconstitution.
Authors: Kopp Hans-Georg , Avecilla Scott T. , Tejada Rafael , Crystal Ronald G. , Hackett Neil R. , Rafii Shahin
Published: April 2020

Accumulating evidence from our lab (Nature Med.2004; 10(1): 64–71) and others have shown that myelosuppression not only results in apoptosis of the hematopoietic cells but also in regression of bone marrow (BM) sinusoidal vessels. Remarkably, we have shown that restoration of hematopoiesis is dependent on the regeneration of BM sinusoidal neo-vessels. However, the mechanism whereby vascular reconstitution is regulated is unknown. Because Tie2/Angiopoietins are critical in the remodeling of neo-vessels, we hypothesized that activation of Tie2 plays a role in regeneration of sinusoidal neo-vessels and restoration of hematopoiesis. To this end, we took advantage of transgenic mice where the Tie2 promoter drives the expression of beta-galactosidase (LacZ) in order to follow the expression of Tie2 in hematopoietic progenitor cells after myelosuppression. Under steady-state conditions, only few LacZ/Tie-2+ cells were localized in the endosteal/osteoblastic region, without any expression within the BM sinusoidal neo-vessels. However, after a myelosuppressive dose of 5-Fluorouracil (5-FU) there was a robust expression of Tie2 expression in the regenerating BM sinusoidal neo-vessels. The expression of Tie2 on the regenerating neo-vasculature reached a maximum at day 10 and reverted back to steady-state by day 20 post 5-FU, the time in which the majority of the neo-vessels were functionally assembled.

Blocking angiopoietin/Tie2 signaling with the soluble decoy-receptor, Tie2Fc, after 5-FU inhibited platelet recovery as well as vascular reconstitution. There was a paradoxical accumulation of megakaryocytes in the bone-marrow during the prolonged thrombocytopenic phase, which we have previously shown to be due to vascular niche disruption and inability of megakaryocytes to release platelets.

To examine the possibility that Tie2+ progenitors can reconstitute BM sinusoids in the recipient mice, lethally irradiated wildtype mice were transplanted with the BM of Tie2-lacZ knock-in mice. After full recovery, the chimeric mice were challenged with a myelosuppressive dose of 5-FU and Tie2+ neo-vessels were detected histologically on day 5, 10, and 14 by LacZ staining. Remarkably, the reconstituted bone marrow showed the presence of Tie2+ vessels, which unambiguously demonstrates the contribution of donor-derived endothelial progenitors to the reconstitution of the regressed vasculature.

Together, our data support the hypothesis that Tie2/angiopoietin signaling is essential for functional regeneration of BM sinusoidal neo-vessels and contributes to the reconstitution of hematopoiesis, specifically to thrombopoiesis. In addition, the use of Tie2-LacZ mice provides an invaluable model to quantify the number of regenerating BM neo-vessels. Angiopoietins may be used in clinical setting in conjunction with other lineage specific cytokines to enhance hematopoiesis after myelosuppression.
Elderly Patients with Early-Unfavorable Stage Hodgkin’s Disease (HD) Have a Poorer Outcome When Treated with Combined Modality Treatment and Extended Field Radiotherapy (EF): A Retrospective Analysis of the German Hodgkin Study Group.
Authors: Engert Andreas , Haverkamp Heinz , Eich Hans T. , Josting Andreas , Pfistner Beate , Diehl Volker
Published: April 2020

Purpose: The HD8 study of the German Hodgkin Study Group (GHSG) demonstrated that involved field (IF) radiotherapy is equally effective when compared with EF radiotherapy after four cycles of chemotherapy (2 x COPP/ABVD). Since there are indications that elderly patients with HD might fare worse depending on the type of treatment applied, we revisited the HD8 data for possible differences between younger and older patients.

Methods and results: A total of 1204 patients were randomised to receive two double cycles of COPP/ABVD and either 30 Gy EF + 10 Gy bulk or 30 Gy IF + 10 Gy bulk. Of these, 98 evaluable patients were older than 60 years and 1038 patients were younger than 60 years. In general, there were more risk factors such as B-symptoms, elevated ESR, and poorer Karnofski index in the elderly group. On the other hand, there were fewer bulky tumours, large mediastinal tumours and a lower number of lymph node areas involved in elderly patients. The toxicity of treatment was more pronounced in elderly patients with 76 of 96 patients experiencing chemotoxicity Grade III or IV (79%) compared with 699 of 1018 (69%) in those younger than 60 years. After a median follow up of 52 months, the 5-year-FFTF was 85% in younger patients and 63% in patients older than 60 years (p <0.001). The 5-year-overall survival was 94% for patients younger than 60 years and 66% for patients older than 60 years (p < 0.001). In addition, patients older than 60 years treated with EF had a trend for worse FFTF and overall survival compared to those receiving IF radiotherapy.

Conclusion: Event-free and overall survival of patients older than 60 years old are worse compared with younger patients. In particular, patients older than 60 years receiving EF radiotherapy had a poorer prognosis.
Comparison of Patients (Pts) Characteristic and Treatment Outcomes in Lymphocyte-Predominant (LPHD) and Classical Hodgkin’s Disease (cHD): A Report from the German Hodgkin Lymphoma Study Group (GHSG).
Authors: Nogova Lucia , Reineke Thorsten , Josting Andreas , Behringer Karolin , Pfistner Beate , Diehl Volker , Engert Andreas
Published: April 2020

Introduction: LPHD differs in histological and clinical presentation from cHD. Treatment of LPHD patients (pts) using standard Hodgkin disease (HD) protocols leads to complete remission (CR) in more than 95% of pts. However, differences in terms of relapse rates, survival and freedom from treatment failure (FFTF) between clinical stages of LPHD and cHD pts were suggested by a recent intergroup analysis. To obtain a more comprehensive picture, we reviewed all LPHD-cases registered in the GHSG database and compared patient characteristics and treatment outcome with cHD pts.

Patients and methods: We retrospectively analysed 8298 HD pts treated within the GHSG trials (HD4 to HD12): 394 LPHD pts and 7904 cHD pts. From 394 LPHD pts 63% were in early stage, 16% in intermediate and 21% in advanced stage of disease. Of the 7904 cHD pts analysed, 22% were in early, 39% in intermediate and 39% in advanced stages. 9% of LPHD pts had B symptoms compared to 40% in cHD pts.

Results: 87.5% LPHD pts reached CR/CRu compared to 81.1% cHD pts. 0.3% LPHD pts developed progressive disease (PD) compared to 3.7% cHD. The relapse rate of LPHD pts was very similar to cHD (6.9%). 0.8% LPHD pts and 3.1% cHD had early relapse, 6.9% LPHD pts and 4.2% cHD pts suffered from late relapse. LPHD pts with late relapse were in intermediate stages (12.5%). In contrast, only 3.9% of cHD pts in intermediate stages had late relapses. There were 2.5% secondary malignancies in LPHD and 3.7% in cHD pts. 4.3% LPHD pts and 8.8% cHD pts died. The FFTF rates for LPHD or cHD pts at a median observation of 41 or 48 months, respectively were as follows: early stages 93% vs. 87%, in intermediate stages 87% vs. 85% and in advanced stages 77% vs. 75%. The OS rates for LPHD or cHD pts were 98% vs. 96% for early stages, 94% vs. 93% for intermediate stages and 91% vs. 87% for advanced stages. The multivariate analysis of prognostic and risk factors will be presented.

Conclusion: cHD pts present more frequently with advanced stages and B symptoms compared to LPHD pts. Comparing LPHD and cHD pts we found differences in treatment outcome in respect of CR/CRu, progressive disease and mortality. Surprisingly, there were no differences in terms of relapses. The majority of late relapses in LPHD pts were in intermediate stages. In contrast to previous reports, our data suggest that relapses in LPHD and cHD pts seem to be comparable and not more frequent. Thus new treatment strategy for LPHD pts in intermediate stages should be considered.
Impact of Course of Therapy on Response Rate and Duration with the Bexxar® Therapeutic Regimen (Tositumomab and Iodine I 131 Tositumomab) and with Chemotherapy.
Authors: Leonard John P. , Zelentz Andrew D. , Vose Julie M. , Kaminski Mark S.
Published: April 2020

Low grade NHL with or without transformation is characterized by recurrence. Initial response rates with chemotx are high, but all patients ultimately relapse, and the response rates and durations of response typically decline with each subsequent course of chemotx. In the pivotal trial of Bexxar (JCO 19:3918 2001) there were significantly more patients with longer durations of response than after their last qualifying chemotx. The aim of the current retrospective study was to evaluate the efficacy of Bexxar and prior chemotx by course of treatment in a larger group of patients. Response rates (RR) and duration of response data were available from the treatment histories of the 230 patients from five clinical studies. Demographics: median age, 56 years; age > 60, 34%; median number of prior regimens, 3 (range 1–13); bone marrow involvement, 48%; bulky disease, 32%; transformed histology 28%; IPI score ≥ 2, 81%; and refractory to their preceding regimen, 80%. The patient-specific dosing regimen for Bexxar was previously described (JCO 19:3918). RR and durations for chemotx used as the 2nd, 3rd, 4th, 5th, 6th and >6th treatment were compared to Bexxar for the same numerical treatment. At each successive treatment course, there was a consistently higher RR and duration of response after Bexxar than after chemotx (Table). The actual differences in RR may be greater, as the data for Bexxar are based on responses observed on at least 2 evaluations >4 weeks apart, as assessed by an independent, blinded Masked Independent Randomized Radiology and Oncology (MIRROR) panel. The reported RR for chemotx are based on Investigator-assessed unconfirmed responses. Of note, the % responding pts in continuous response at ≥ five year after receiving Bexxar for the 2nd, 3rd, 4th, 5th, 6th and >6th relapse were 41%, 21%, 25%, 35%, 36 and 9%, respectively. While RR decreased with number of prior therapies, the durations of response and percent in continuous response after Bexxar were substantial and consistent across the range of the number of prior therapies.

Treatment/Regimen Number Chemotx Bexxar n Response Rate (Unconfirmed) Median Duration of Response (months) n Response Rate (Confirmed) Median Duration of Response (months) 2 183 51% 8.0 27 74% 14.4 3 145 52% 5.0 44 64% 47.2 4 98 45% 5.0 54 52% 10.8 5 45 40% 4.0 48 54% 11.0 6 33 36% 4.0 21 52% 27.0 7 or more 48 19% 3.5 36 42% 9.6
Delayed Severe Neutropenia after Rituximab Therapy in 18 among 293 Patients with B-Cell Malignancies Treated in a Single Institution. an Adverse Event of Unknown Mechanism.
Authors: Belhadj Karim , El Gnaoui Taoufik , Doghmi Kamal , Joly Bertrand , Lee Ketty , Plonquet Anne , Jouault Hélène , Gourlain Karine , Delfau-Larue Marie-Hélène , André Chantal , Reyes Felix , Haioun Corinne
Published: April 2020

The characteristics of severe neutropenia with delayed onset following administration of rituximab were evaluated in 293 consecutively patients treated for B-cell malignancies between Oct. 1996 and Feb. 2004. Eighteen episodes of severe neutropenia were identified between 2 to 36 weeks after rituximab, delivered alone in 4 cases or combined to chemotherapy in 14 other cases. At the time of occurrence of neutropenia, 10 patients had completed treatment and were in complete remission and 8 were still on therapy. All patients had a normal neutrophil count before rituximab treatment. In 4 cases, neutropenia was complicated by fever requiring hospitalisation. Among the18 patients [diffuse large B-cell lymphoma: 12, follicular lymphoma: 3, mantle cell lymphoma: 1, post transplant lymphoproliferative disorder: 1, chronic lymphocytic leukemia (CLL): 1], 8 had previouly been treated with high-dose therapy followed by autologous stem-cell transplantation. None of the known causes of neutropenia were found. Neutropenia was associated with selective marrow depletion of neutrophil precursors in all episodes. The peripheral blood lymphocyte subset repartition was documented in 16 cases; a severe B lymphopenia was observed in all except one - patient with CLL - and CD4+ lymphopenia was seen in 13. T-cell clonality was studied in 8 cases and was positive in 5. Parvovirus B19 DNA was not detected in the 11 tested cases. Tests for antineutrophil antibodies were positive in 5 cases; no hemolytic anemia, autoimmune thrombocytopenia or large granular natural killer lymphocytosis were observed. Filgrastim was given to 4 patients only. The median time of neutrophil recovery for the entire group was 14 days (range: 2–180).

The mechanism of this rare event already reported (Saikia et al. Ann. Oncol 2002, Voog et al. NEJM 2003, Chaiwatanatorn et al. BJH 2003, Lemieux et al. BMT 2004) remains to be established. Since it seems to be more frequent in patients who have received extensive prior chemotherapy (8/18), it is tempting to speculate that cumulative immunosuppression might favor the development of such an event.
Treatment of Complete DiGeorge Syndrome by Repeat Transfusions of Blood Lymphocytes from an HLA-Identical Sibling Donor.
Authors: Hoenig Manfred , Schulz Ansgar , Schuetz Catharina , Debatin Klaus-Michael , Friedrich Wilhelm
Published: April 2020

DiGeorge syndrome (DGS) is a congenital disorder characterized by facial dysmorphies, hypoparathyroidism, cardio-vascular malformations and variable T-cell deficiency due to thymic hypoplasia. We present a male infant with complete absence of T-cells and thymus successfully treated by repeat blood lymphocyte transfusions. According to previous experience of bone marrow transplantation in a similar patient, T-cell reconstitution is possible by peripheral expansion of mature donor cells, independent of stem cell engraftment. Our patient received at the age of four months an initial transfusion of blood lymphocytes from his HLA-identical brother, containing 1x108 T-cells/ kg. Ten days later he developed mild cutaneous GvHD, which resolved spontaneously without immunosuppressive therapy. Lymphocyte transfusions were repeated three times at 4 weeks intervals without reappearance of GvHD. T-cells developed within two months after the first transfusion and since then remain at levels between 500–1000/μl with a proportion of 25% disclosing a CD45RA+ phenotype. In chimerism analysis T-cells are exclusively of donor origin. The T-cell receptor repertoire is similarly diverse as in the donor. After more than 2 years of follow up the boy is not suffering from severe infections and shows no signs of autoimmune disease. Following immunization, antibody titres normalized. Conclusion: In infants with complete DGS transfusions of blood lymphocytes from a matched family donor may lead to long-lasting engraftment of functional T cells and stable chimerism and represents a potentially helpful therapeutic option.
UNC13D and PRF1 Mutations in Childhood Patients with Hemophagocytic Lymphohistiocytosis.
Authors: Stadt Udo zur , Beutel Karin , Oyen Florian , Diler A. Sarper , Kabisch Hartmut , Schneppenheim Reinhard , Hennies Hans-Christian , Janka Gritta
Published: April 2020

Introduction: Familial Hemophagocytic Lymphohistiocytosis (FHLH) is an autosomal recessive disease affecting young children. It manifests itself as a severe hyperinflammatory syndrome with activated macrophages and T-lymphocytes. Mutations in Perforin (PRF1) are responsible for FHL-2 in about 15 to 50 % of the patients. Defective granule exocytosis due to mutations in UNC13D have recently been described as the genetic defect underlying FHL-3. Both types are phenotypically not distinguishable from one another.

Patients and Methods: Genetic analysis was performed in either the 2 or the 32 exons of PRF1 and UNC-13D, respectively, in 61 patients with hemophagocytic lymphohistiocytosis (HLH) of different geographic origin (28 from Germany, 24 from Turkey and 9 from other countries).

Results: We identified mutations in 28 samples investigated (18 in PRF1, 10 in UNC-13D). Besides several known mutations novel deletions, missense and nonsense mutations were detected in both genes scattering the whole coding regions. The Trp374stop mutation (11/13 patients with a PRF1 mutation from Turkey) was the only single mutation repeatedly observed in several patients with a common geographic origin. In HLH patients from Germany, 7/28 patients showed homozygous or compound heterozygous mutations in either PRF1 (3) or UNC13D (4). In 24 HLH patients from Turkey 13 patients with a mutation in PRF1 and 4 patients with a mutation in UNC13D were identified. In addition, four out of 9 patients with a different geographic origin showed mutations in one of this genes. In 10 patients only one heterozygous mutation in UNC13D was detected so far. In these cases, control samples or regulatory regions of the gene have to be analysed to ascertain the relevance of the underlying mutation.

Defect Turkey German Other All UNC13D 4 4 2 10 PRF1 13 3 2 18 Unknown 7 21 5 33

Discussion: Overall, UNC-13D and PRF1 mutations were detectable in 45% of the cases. Our results indicate that FHL-2 and FHL-3 account for more than 60% of the HLH cases of Turkish origin with only a small number involving UNC-13D. In contrast, gene defects were identified in only 25% of the patients with a German ancestry. This work was supported by the Foerdergemeinschaft Kinderkrebszentrum Hamburg e. V.
The Significant Decrease of Recent Thymic Output Function in Patients with Benzene-Poisoned Aplastic Anemia.
Authors: Li Yangqiu , Han Sufang , Yang Lijian , Chen Shaohua , Zhou Yubing , Chen Jiayu , Liu WeiWei
Published: April 2020

It has been known for many years that benzene causes hematotoxicity, as well as the toxicity in lymphopoiesis, and it associated with aplastic anemia and leukemia. The aim of the present study was to investigate the level of T-cell receptor excision DNA circles (TRECs) within peripheral blood mononuclear cells (PBMCs) in patients with benzene-poisoned aplastic anemia (AA), thereby to evaluate the content of naive T cells and the recent thymic output function. Quantitative detection of TRECs in DNA of PBMCs from 16 normal individuals and 7 cases with benzene-poisoned AA was preformed by real-time PCR using TaqMan technique. The results showed that TRECs level was 6.69±4.79/1000 PBMCs in normal individuals, it was significant decrease in patients with benzene-poisoned AA (2.24±1.57/1000 PBMCs, p<0.05). The TRECs levels in 2 cases with benzene-poisoned AA were followed up in different time points up to 55 weeks. The TRECs level was persistent decrease after diagnosis of benzene-poisoned and leaving the benzene exposure workplaces and undergoing clinic specific treatment, even if peripheral blood cell counts were became normal levels. The TRECs levels were 1.35±0.87/1000 PBMCs (at 5 time points) and 0.61±0.45/1000 PBMCs (at 4 time pionts) in two cases respectively. The results indicated that the recent thymic output function was remarkable decrease in patients with benzene-poisoned AA. It may obviously damage the T cell immune function in benzene poisoning.
Proliferation of CD8+ T-Cells Specific for CMV and EBV in Response to TCR Mediated Recognition of Cognate Antigen Is Inhibited by Imatinib in a Dose-Dependent Manner.
Authors: Seggewiss Ruth , Lore Karin , Greiner Elisabeth , Magnusson Magnus K. , Price David A. , Douek Daniel C. , Dunbar Cynthia E. , Wiestner Adrian
Published: April 2020

We and others have shown that the tyrosine kinase inhibitor imatinib (STI571, Gleevec®) inhibits T-cell proliferation and activation at concentrations achieved in vivo. At 10μM, imatinib inhibited T-cell receptor (TCR)-mediated proliferation of purified peripheral blood T-cells almost completely. Up-regulation of the activation markers CD25 and CD69 at 24h in response to TCR cross-linking was suppressed by imatinib at a mean IC50 of 5.4μM and 7.3μM, respectively and IL-2 production was also severely impaired. However, these assays may not fully reflect the response to clinical relevant antigens. Therefore, we chose to investigate the antigen-triggered proliferation of memory CD8+ T-cells specific for immunodominant CMV and EBV HLA-A2 peptide epitopes. We used HLA-peptide tetramers to identify healthy blood donors with detectable CMV- or EBV-specific CD8+ T-cell populations. Purified T-cells from these donors were then stimulated with the CMV peptide pp65495–503 or the EBV peptide BMFLI259–267. Antigen-induced proliferation was measured by dilution of the vital dye CFSE over a period of 4 or 8 days. The magnitude of the virusspecific CD8+ T-cell population ranged from 0.5 % to 7.1% of CD8+ T-cells for CMV and from 0.05% to 0.35% of CD8+ T-cells for EBV. Antigen-specific CD8+ T-cells from all 10 donors studied proliferated in response to the CMV peptide. In 8 from 10 donors, imatinib reduced CMV peptide induced proliferation. With increasing imatinib concentrations (range: 5 – 10μM), we observed dose dependent reduction of both the number of cells undergoing cell division and the average number of divisions completed per cell. Comparable inhibition of specific T-cell proliferation in response to the EBV-derived peptide was observed in two donors. Immunoblots demonstrated that imatinib substantially reduced tyrosine phosphorylation of ZAP70 and LAT in response to TCR-mediated activation in Jurkat T-cells. Sequence comparisons of all 90 tyrosine kinase genes in the human genome for homology in the ATP binding pocket identified Lck, which is required for ZAP70 activation, as a likely target for imatinib. Our results indicate that imatinib may interfere with clinically important T-cell effector functions. As concentrations sufficient for half-maximal inhibition of TCR signalling are achieved in vivo, imatinib could increase the risk of opportunistic infections and impact on GVH and GVL reactions post-transplantation especially when used in conjuction with other immunosuppressive agents. Therefore, close monitoring of patients on imatinib for CMV reactivation or EBV-induced lymphoproliferative diseases, especially in stem cell transplant recipients, appears warranted.
Patients with Lymphoma May Harbor Mutations of the Perforin Gene.
Authors: Clementi Rita , Locatelli Franco , Dupre Loic , Garaventa Alberto , Emmi Lorenzo , Bregni Marco , Cefalo Graziella , Moretta Antonia , Danesino Cesare , Comis Margherita , Pession Andrea , Ramenghi Ugo , Maccario Rita , Arico’ Maurizio , Roncarolo Maria Grazia
Published: April 2020

Perforin (PRF), a pore-forming molecule expressed in granules of cytotoxic effector lymphocytes, has been implicated in T and NK cell-mediated immune surveillance against viral infections and tumors. Indeed, increased lymphoma incidence has been associated with PRF deficiency in mice. In view of these experimental findings, we reasoned that also some patients with lymphomas might harbor mutations of the PRF gene and thus we investigated the presence of mutations of the gene in a group of lymphoma patients with pre-defined clinical characteristics. Bi-allelic mutations of the PRF gene were found in 4 out of the 30 patients examined. A female (19 y.) had been successfully cured of her T-cell lymphoblastic lymphoma, when her brother(22 y), 2 years later, developed HLH. The 2 siblings shared the same mutations. The first one, (272 C>T), changes arginine at position 91 into valine; the second one (1122 G>A), changes tryptophan at position 374 into a stop codon. The second patient (7 y), with EBV-positive Hodgkin lymphoma,three years later, developed a large B-cell non-Hodgkin lymphoma. Two mutations were identified: the first one, (1304 C>T), changes threonine at position 435 into methionine. The second one, (1349C>T), changes threonine at position 450 to methionine. The third patient (7 y), a female, presented, with multiple reddish non-ulcerated subcutaneous nodules and B-symptoms. Two surgical lesion biopsies were performed and a rare form of subcutaneous, panniculitis-like, T-cell lymphoma was diagnosed. Sequencing of the PRF gene disclosed the following 2 mutations: the first one, (272 C>T), changes arginine at position 91 into valine; the second one, (1262 T>G), changes phenylalanine at position 421 into cysteine. An female (18 y)presented with fever, malaise and massive hepato-splenomegaly. A peripheral T-cell lymphoma, associated with bone marrow infiltration, was diagnosed and 6 months after diagnosis, she was transplanted from an HLA-identical sibling. PRF gene sequencing showed two novel mutations: the first one (g914a) changes at position 305 glycine in aspartic acid, the second one (c1066a) changes arginine in tryptophan at position 356. Present data, point to a more complex disregulation of the immune system when PRF is absent, which may be associated with quite different clinical presentations indicating that the resulting effect of PRFmutations is likely to be more complex than previously anticipated and may vary among individuals, possibly depending on additional genetic and/or environmental factors.