bluuebunny/crossref_metadata_2025 huggingface dataset:
CMV-Seropositive Patients Allografted with a CMV-Seronegative Donor Show Delayed or Missing CMV-Specific T-Cell Immune Response and Are at Higher Risk for CMV-Viremia and CMV-Disease.
Authors: Ganepola Susanne , Gentilini Chiara , Hilbers Urte , Hartung Goetz , Lange Thoralf , Hofmann Joerg , Liebert Uwe G. , Hammer Markus , Engelmann Elisabeth , Niederwieser Dietger , Thiel Eckhard , Uharek Lutz
Published: April 2020

The human cytomegalovirus (CMV) is an important cause for mortality and morbidity after allogeneic stem cell transplantation (allo SCT) especially in patients without a CMV-specific T cell response. CMV seropositive patients allografted with a CMV seronegative donor do not rise up specific CMV-T cell immunity posttransplant and are therefore at great risk for CMV viremia and disease. In a prospective manner, we analyzed blood samples of 38 HLA-A2+ patients with different haematological malignancies for CMV specific T cells.

Methods: Frequency of T cells with reactivity against the pp65-peptide (NLVPMVATV) was assessed by ELISPOT assay in 38 patients at defined time points after allo SCT. In patients with high CMV specific T cell frequencies, the T cell phenotype was determined by flow cytometry. Surveillance of CMV viremia was carried out by routine PCR-technique or pp65 Ag staining.

Results: In high-risk patients (donor-/recipient+) viremia was observed in 7/9 patients, 3/7 developed clinical severe CMV-disease. In this group, only one patient presented a weak CMV-specific T cell response in the first year after transplantation, although CMV-specific T cells were demonstrated in 5/9 patients before transplantation. In contrast, 64% (11/17) of the CMV seropositive patients having had a CMV seropositive donor showed CMV-specific T cells around day 30. Their T cell frequencies remained stable at a relative high level during the whole time of our investigation period and no CMV disease was observed. CMV seronegative patients allografted with either a CMV seronegative or seropositive donor also remained disease-free. CMV specific T cells were detectable in 2/7 patients of the d+/r− group. FACS analysis revealed that most of the responding cells were of the activated effector phenotype CD8+/CD45RA+/HLA-DR+ with a low expression of CCR7 and CD27.

Conclusion: CMV-seropositive patients receiving graft from a seronegative donor are at a high risk and therefore prone for CMV-viremia and -disease. The development of CMV-specific T cells i.e. immune reconstitution in high risk patients remains delayed or is completely missing during the first year post transplant. In contrast, seropositive recipients grafted with a seropositive donor develop a durable T cell response within 3–4 weeks and show no viremia at all. New strategies as donor vaccination and adoptive T cell transfer are warranted to prevent CMV-disease in CMV seropositive patients for whom only a seronegative donor can be found.
CMV Reactivation after Allogeneic Stem Cell Transplantation Is Associated with a Preponderance of Circulating Late-Stage CMV-Specific CD8+ T Cells.
Authors: Komanduri Krishna V. , St. John Lisa S. , Shpall Elizabeth J. , Champlin Richard E. , Molldrem Jeffrey J. , Ozdemir Evren
Published: April 2020

CMV reactivation remains a major cause of morbidity after after allogeneic stem cell transplantation. Using HLA-peptide tetramer staining and cytokine flow cytometry, we previously demonstrated that higher CD4+ and CD8+ T cells specific for CMV were found in the peripheral circulation of individuals who experienced CMV reactivation, but that more of these cells were dysfunctional in subjects experiencing viral reactivation. Recent studies have examined the role of maturation status of virus-specific T cells in the setting of of cleared, persistent and progressive viral infections. CD57 is a marker of replicative senescence that is expressed on a subset of human CD8+ memory T cells that generally lacks CD28 expression, and that is characterized by downregulation of the naïve and central memory T cell markers CD45RA and CCR7. To examine whether CMV reactivation after SCT was associated with late-stage differentiation of CMV-specific CD8+ T cells specific for viral proteins, we first examined the fraction of CD57-expressing CD8+ T cells in patients stratified by the occurrence of post-SCT viral reactivation. In 87 patients examined with HLA-peptide tetramer staining at approximately 3 months after allogeneic SCT, we found that individuals experiencing CMV reactivation within the first 100 days after SCT had significantly higher absolute numbers of circulating CD57+ tetramer-stained CD8+ T cells specific for CMV than those who did not experience viral reactivation (mean 11.8 vs. 1.6 CD57+ tetramer-stained cells/μl, median 3.3 vs. 0.08 cells/μl, p<0.0001). In individuals in whom >75% of tetramer-stained CD8+ T cells were CD57+, the incidence of CMV reactivation was 83%, in contrast to 52% of others. In other experiments, we found that CD57+ tetramerstained CD8+ T cells failed to expand following co-culture with autologous monocyte-derived dendritic cells pulsed with CMV peptide pools, confirming that CD57+ CMV-specific CD8+ T cells may have impaired proliferative capacity. Taken together, these results suggest that late-stage differentiation of memory CD8+ T cells may be indicative of an exhausted antigen-specific immune response, and that optimal post-SCT immune restoration should consider the differentiation status and not simply the numbers of antigen-specific T cells targeted.

Figure Figure
The Platelet-Derived Growth Factor Receptor beta Fuses to Two Distinct Loci at 3p21 in Imatinib Responsive Chronic Eosinophilic Leukemia.
Authors: Curtis Claire , Apperley Jane F. , Dang Raymond , Jeng Michael , Gotlib Jason , Cross Nicholas C.P. , Grand Francis H.
Published: April 2020

We have identified three patients (2 adults, one infant) who presented with BCR-ABL negative eosinophilic myeloproliferative disorders. Cytogenetic analysis revealed a t(1;3;5)(p36;p21;q33) for case 1 and a t(3;5)(p21–25;q31–35) for cases 2 and 3. Two-color fluorescence in situ hybridization (FISH) using differentially labelled probes flanking PDGFRB indicated that this gene was disrupted in all three cases. 5′ rapid amplification of cDNA ends (5′RACE) for case 1 identified an in-frame mRNA fusion of exon 9 of the WDR48 gene at 3p21 to exon 12 of PDGFRB. The chimeric mRNA is predicted to encode a 872 amino acid fusion protein that retains the amino terminal WD repeat region of WDR48 fused to the transmembrane and intracellular tyrosine kinase domains of PDGFRbeta. Cases 2 and 3 were negative for the WDR48-PDGFRB fusion mRNA by RT-PCR using several combinations of primers. 5′RACE PCR from case 2 RNA identified a fusion involving a second 3p21 gene: GOLGA4 exon 11 was fused in-frame to exon 11 of PDGFRB. Exactly the same fusion was found in case 3. The predicted 991 amino acid protein included the amino terminal coiled-coil domain of GOLGA4 fused to the transmembrane and intracellular tyrosine kinase domains of PDGFRbeta. Interestingly, both WDR48 and GOLGA4 are involved in endocytic shuttling pathways. The presence of all fusions was confirmed by RT-PCR and identification of the genomic breakpoints. Imatinib, a known inhibitor of PDGFRbeta, selectively blocked the growth of patient CFU-GM for case 2. Following the identification of PDGFRB rearrangements, all three patients were treated with imatinib. Case 1 was in transformation, but responded rapidly to minimal doses of imatinib (800mg daily for 4 days) with complete cytogenetic remission but remained pancytopenic. Blast crisis recurred 8 months later, responded similarly to 3 days of imatinib, but the patient died 2 months later of invasive fungal infection. Case 2 responded clinically and remains in sustained cytogenetic and molecular remission (nested RT-PCR negative for GOLGA4-PDGFRB). Case 3 (a 13 month old boy) had a complete hematologic response to 50mg/day imatinib but the t(3;5) was still seen in 40% of metaphases at 3 months. We conclude that PDGFRB fuses to diverse partner genes to give rise to atypical MPDs. Although very rare, identification of these fusions is critical for proper management of affected individuals.
Activation of HIV-1-Specific CD8+ and CD4+ Autologous Memory T-Cells by Dendritic Cells and B-Cells Electroporated with mRNA Encoding Consensus or Autologous HIV-1 Proteins.
Authors: Berneman Zwi N. , Van Gulck Ellen R.A. , Van den Bosch Glenn , Ponsaerts Peter , Heyndrickx Leo , Moerman Filip , De Roo Ann , Lenjou Marc , Nijs Griet , Van Bockstaele Dirk R. , Van Tendeloo Viggo F.I. , Maranon Concepcion , Hosmalin Anne , Vanham Guido
Published: April 2020

Human immunodeficiency virus type 1 (HIV-1) infection is characterized by dysfunction of HIV-1-specific T-lymphocytes. In order to suppress the virus and delay evolution to AIDS, antigen-loaded antigen-presenting cells (eg. dendritic cells (DC), B-lymphocytes) might be useful to boost and broaden HIV-1-specific T-cell responses. Monocyte-derived DC from untreated HIV-1-infected patients were electroporated with codon-optimized (“humanized”) mRNA coding for consensus HxB-2 (hHXB-2) Gag protein. These DC elicited a strong HIV-1 Gag-specific interferon (IFN)-γ response by an HLA-A2-restricted CD8+ T-cell line. Moreover, hHXB-2 gag mRNA-electroporated DC also triggered IFN-γ secretion by autologous peripheral blood mononuclear cells (PBMC), CD8+ T-cells and CD4+ T-cells from all patients tested. Similar observations were made with CD40-activated cultured autologous B-cells (from HIV-1-seropositive patients) electroporated with hHXB-2 gag mRNA. Gag mRNA-electroporated, but not mock-electroporated, DC or B-cells secreted Gag protein. Next, a novel strategy was developed, using autologous virus sequences. Proviral DNA was amplified by polymerase chain reaction (PCR) from PBMC and viral cDNA was amplified by reverse transcriptase PCR (RT-PCR) from plasma virus. Proviral and viral mRNA were then obtained by in vitro transcription of proviral DNA and plasma viral cDNA, respectively. Significant specific IFN-γ T-cell responses were induced in all patients tested by DC electroporated with patients’ autologous proviral and plasma viral mRNA, coding for Gag or Env. The stimulatory effect was seen on PBMC, CD8+ T-cells and CD4+ T-cells, demonstrating both major histocompatibility complex (MHC) class I and MHC class II antigen presentation. Moreover, a significant interleukin (IL)-2 T-cell response was induced by DC electroporated with hHxB-2 or proviral gag mRNA. Sequence analysis in 4 randomly chosen patients showed that they were infected by 4 different subtypes. In a heteroduplex mobility assay (HMA) up to 50% of the cloned amplified sequences exhibited a differential migration pattern; by sequencing a high degree of variation was demonstrated, particularly between clones derived from proviral DNA and plasma viral cDNA, with mutations in an immunodominant epitope (Gag) or mutations and deletions in non-immunodominant epitopes (Env). The stimulatory effect of autologous DC electroporated with autologous viral sequences opens a major perspective for the development of patient-specific immunotherapy for HIV-1 disease, that might be necessary to control the virus, in view of the major inter-patient and intra-patient sequence variability.
Quantitative Assessment of Molecular Remission Following Autologous Stem Cell Transplantation Predicts Long Term Remission in Mantle Cell Lymphoma.
Authors: Pott Christiane , Schrader Carsten , Gesk Stefan , Harder Lana , Tiemann Markus , Raff Thorsten , Brüggemann Monika , Ritgen Matthias , Unterhalt Michael , Dreyling Martin , Hiddemann Wolfgang , Siebert Reiner , Dreger Peter , Kneba Michael
Published: April 2020

Mantle cell lymphoma (MCL) is characterized by a poor response to cytostatic therapy and a short survival with a median of less than 3 years. High-dose chemotherapy followed by autologous peripheral stem cell transplantation (ASCT) provides a potential curative option for the treatment of younger patients. The prognostic relevance of minimal residual disease (MRD) detection after intensive conventional treatment and high dose therapy has been proven for follicular lymphoma but is still under debate in MCL.

Aim of this study was the quantitative evaluation of MRD and the evaluation of its prognostic impact on progression free survival and long-term remission in patients with MCL after ASCT.

Quantitative Real-Time PCR for clonal IGH rearrangements (IGH-RQ-PCR) was performed in 29 patients with MCL treated with CHOP-like induction, stem-cell mobilisation with DexaBEAM. and subsequent ASCT. Quantitative MRD assessment was performed prior and during treatment as well as 3,6 and 12 months after PBSCT by IGH-RQ-PCR. 14/27 patients evaluable for MRD after ASCT achieved a complete clinical and molecular remission, whereas 13 patients had detectable MRD within the first year after ASCT. Molecular remission after ASCT was strongly predictive for improved outcome with a median PFS of 95 months in the MRD negative group compared to 21 months in the MRD positive group (p<0.0001). Overall survival differed significantly with 55.8 months in the MRD positive group, whereas median OS of the MRD negative group has not been reached (p<0.003). In multivariate analysis, molecular remission and bulky disease were independent prognostic factors for PFS (p=0.0007 and p=0.0210 respectively). Quantitative MRD measured in the stem cell products of 28 patients was not predictive for achieving molecular remission. We conclude that sequential quantitative monitoring of residual disease after ASCT is a powerful indicator for treatment outcome in MCL and defines subgroups of patients with a significantly different prognosis.
VEBEP Regimen and Highly Active Antiretroviral Therapy (HAART) in Patients (pts) with HD and HIV Infection (HD-HIV).
Authors: Spina Michele , Rossi Giuseppe , Antinori Andrea , Allione Bernardino , Fasan Marco , Rizzardini Giuliano , Tirelli Umberto
Published: April 2020

Background: The outcome of pts with HD-HIV is still poor, because the duration of complete remission (CR) is short. To improve the prognosis of HD-HIV, a feasibility study with the VEBEP regimen and radiotherapy and concomitant HAART was started in previously untreated HD-HIV pts.

Methods: CT included epirubicin 30 mg/m2/day (days 1–3), cyclophosphamide 1000 mg/m2 (day 1), vinorelbine 25 mg/m2 (day 1), bleomycin 10 mg/m2 (day 3) and prednisone 100 mg/m2/day (days 1–3).

Results: Since September 2001, 28 pts have been enrolled. The median age was 39 yrs. The median CD4+ cell count at entry was 256/mm3 (range 44–589) and 15 pts had a detectable HIV viral load (median 9402 copies/mm3, range 89->500000). Stage III and IV disease was present in 19/28 (68%) pts. Histologic subtypes were: MC 75%, NS 14%, not determined 7%, LP 4%. One toxic death, due to hepatic failure in a HCV positive pt was observed. An absolute neutrophil count <500 was noted in 12/28 pts (43%). Grade 3–4 anemia was observed in 8/28 pts (29%) and severe thrombocytopenia in 5/28 pts (18%). Nine pts (32%) had febrile neutropenia with 6 documented infections in 5 pts (one PCP, one cerebral toxoplasmosis, one bacterial sepsis, one bacterial pneumonia, one salmonellosis, one varicella). A grade 2–3 mucositis was observed in 6/28 pts (21%). CR was obtained in 21/28 pts (75%) and PR in 2/28 pts (7%). With a median follow up of 24 months, only 2 pts have relapsed (9%). OS and TTF at 2 years are 86% and 68%, respectively.

Conclusions: Our preliminary data demonstrate that VEBEP regimen in combination with HAART is feasible and active in pts with HD-HIV. This study was supported by ISS grants.
Pretreatment Cytogenetic Abnormalities Are Predictive of Induction Success, Cumulative Incidence of Relapse and Overall Survival in Patients >60 Years of Age with Newly Diagnosed Acute Myeloid Leukemia.
Authors: Schlenk Richard F. , Kayser Sabine , Morhardt Martina , Döhner Konstanze , Döhner Hartmut , Fröhling Stefan
Published: April 2020

Purpose: Karyotype at diagnosis provides the most important prognostic information in younger adults with acute myeloid leukemia (AML). However, there are few data available looking in particular at patients (pts.) above 60 years of age. We prospectively analyzed 361 elderly pts. with newly diagnosed AML. All pts. were treated within the AMLHD98B treatment trial and received intensive induction and consolidation therapy. Pts. exhibiting a t(15;17) received an age-adjusted AIDA-regimen. Median follow-up time was 48 months. The median age was 67 years (range 60–85 years).

Results: 160 pts. had a normal karyotype (44%); 48 pts. (13%) exhibited the balanced translocations t(8;21) (n=12), inv(16) (n=14), t(15;17) (n=11), or t(11q23) (n=11); in the absence of these balanced translocations, 73 pts. exhibited a single aberration, 179 pts. two aberrations, and 61 pts. a complex karyotype (≥3 aberrations; including 44 pts. with 5 or more aberrations). Analyses were normalized to the complete remission (CR) rate (52%), cumulative incidence of relapse (CIR) (77%) and overall survival (OS) (13%) after 4 years of pts. with normal karyotype. Pts. exhibiting a t(15;17) showed a significantly better CIR (29%) and OS (55%), whereas pts. with the other balanced translocations [t(8;21), inv(16)/t(16;16) and t(11q23)] did not differ from pts. with normal karyotype. The limited backward selected Cox-model for OS [t(15;17) excluded] revealed two risk groups: standard-risk [normal karyotype, t(8;21), inv(16), t(11q23), +8 and +11 in absence of a complex karyoytpe] and high-risk [all other aberrations]. The CR rates were 56% and 18%, and the OS-rates after 4 years for the standard- (n=223) and the high-risk group (n=127) were 15% and 0%, respectively. The MRC risk classification system for patients >55 years applied to our patients revealed CR- and OS-rates after 4 years of 73% and 19%, 47% and 12%, as well as 7% and 0% for the low (n=26), intermediate (n=282) and high risk groups (n=44), respectively [t(15;17) excluded]. In conclusion, our risk classification system identified a large high-risk group (36%) of elderly patients with AML who did not benefit from intensive chemotherapy.
Bmi-1 Expression Is Useful To Design Therapy and To Predict Prognosis in Patients with Acute Myeloid Leukemia.
Authors: Chowdhury Moniruddin , Mihara Keichiro , Nakaju Nanae , Fukumoto-Hidani Sachiko , Takihara Yoshihiro , Kimura Akiro
Published: April 2020

Since prognosis of patients with acute myeloid leukemia (AML) is highly variable even in a single subpopulation in FAB classification, it would be useful to find prognostic molecular markers for AML. Thus, we investigated Bmi-1 expression in AML cells by flow cytometry and analyzed whether it predicts prognosis in AML patients and further it is helpful to choose therapies in the modalities of treatment options, because it is known to be required for self-renewal mechanism of leukemic stem cells. Bmi-1 expression in bone marrow or peripheral blood cells was analyzed in 49 patients with AML (M0(n=5), M1(n=7), M2(n=6), M3(n=5), M4(n=8), M5(n=5), M6(n=1)), granulocytic sarcoma(n=1), MDS-AML (n=9), and secondary AML(n=2). Freshly isolated AML cells were stained with a PE-conjugated anti-CD34-antibody followed by fixation and then with anti-Bmi-1-antibody-FITC. All of patients with low Bmi-1 positivity (<35%, n=11) except for de novo AML(M0) entered in complete remission (CR) with single induction chemotherapy(n=5) and accordingly had better overall survival, even though lower dose of chemotherapy (60% of standard dose) was given (n=3). Alternatively, patients with higher percentage of Bmi-1 expression in AML cells (> 70%, n=19) except for AML(M3) progressed to death within two years, unless they were treated with highly intensive therapy such as high dose AraC or allogeneic stem cell transplantation (n=3). Patients with intermediate degree of Bmi-1 expression (35–60%, n=5) responded to standard intensity of chemotherapy (n=2) and are alive for more than two years. Interestingly, patients with MDS-AML (n=9) had high Bmi-1 expression (79%) and all of them have died within 20 months. Binary logistic regression model showed that significant correlation was found among survival status as dependent variable, Bmi-1, and treatment intensity as independent variable (p = 0.004). On the other hand, Univariate analysis did not reveal any relation of Bmi-1 expression to karyotype, age, WBC count, or FAB subtype. In conclusion, Bmi-1 expression could be an independent prognostic marker and useful tool to design therapy for the AML patients.
Defective Cell Cycle Induction by IL-2 in Naïve T-Cells Antigen-Stimulated in the Presence of Refractory T-Lymphocytes.
Authors: Inaba Hiroto , Geiger Terrence L.
Published: April 2020

CD4+ T-cells enter a transient refractory period after stimulation. Upon re-stimulation the refractory cells fail to produce IL-2 and show diminished proliferation. We previously demonstrated that refractory T-cells can also, like anergic and CD4+CD25+ regulatory T-cells, suppress in trans the proliferation of antigen-stimulated naïve T-cells. The suppressed T-cells upregulate high affinity IL-2R but do not produce IL-2, potentially explaining the proliferation failure. We further analyze here suppression mediated by refractory T-cells and find that it cannot be exclusively explained by IL-2 deprivation. Depletion of IL-2 from stimulated naïve T-cells with a blocking antibody results in a pattern of proliferation suppression and CD25 upregulation distinct from that caused by refractory T-cells. Further, supplementation of refractory-naïve cell co-cultures with exogenous IL-2 fails to alleviate refractory cell-mediated suppression, demonstrating that IL-2 deficiency is not exclusively responsible for it. Proximal IL-2 signaling through STAT5 and AKT is intact. However, refractory cell-co-cultured T-cells fail to upregulate cyclins and c-myc and incompletely downregulate p27kip1 in response to IL-2. IL-2 signaling is not fully disabled as IL-2 upregulates the anti-apoptotic protein Bcl-xL to control levels. This upregulation correlates with enhanced survival of co-cultured T-cells placed in IL-2 when compared with cells cultured without IL-2. Thus refractory T-cells are able to suppress naïve T-cell proliferative responses both by blocking IL-2 production and by blocking the mitogenic but not anti-apoptotic effects of IL-2. These results have implications for how activation-refractory T-cells may influence nascent immune responses.
Human GITR Ligand Is Expressed on Tumor Cells and Reduces Cytokine Production and Cellular Cytotoxicity of NK Cells Identified to Express GITR.
Authors: Baltz Katrin M. , Krusch Matthias , Markus Radsak P. , Mayer Frank , Kanz Lothar , Salih Helmut R.
Published: April 2020

Members of the tumor necrosis factor (TNF) superfamily mediate multiple cellular functions including cellular proliferation, differentiation, and cell death. Human Glucocorticoid-induced TNF Receptor (GITR) has been shown to be expressed on T cells, is upregulated following activation and mediates costimulatory signals. The human GITR ligand (GITRL) has been reported to be expressed on antigen presenting cells and various healthy nonlymphoid tissues including small intestine, ovary, testis, kidney and endothelial cells. We analyzed multiple tumor cell lines of hematopoietic and epithelial origin as well as of germ cell lineage and various gliomas by RT-PCR and FACS analysis. Both GITRL m-RNA and protein are expressed in various carcinomas, gliomas and tumor cells of germ cell lineage, but not in hematopoietic tumor cells. Furthermore, we demonstrate that human NK cells constitutively express low levels of GITR, and this expression is upregulated following activation by, e.g., IL-2 or IL-15 as detected by quantitative PCR and FACS analysis. To address the functional interaction of GITRL with its receptor on NK cells, we generated a GITRL-IgG fusion protein (GITRL-Ig). Stimulation of activated NK cells with GITRL-Ig lead to significantly reduced IFN-g production of NK cells as measured by ELISA. Similarly, a significant reduction of IFN-g release was observed following coculture of GITR expressing NK cells with C1R cells transfected with GITRL but not with the respective mock transfectants. Furthermore, ligation of GITR on NK cells lead to significantly decreased killing of target cells as demonstrated by cellular cytotoxicity assays. Taken together, our data demonstrate that GITR not only plays an important role in adaptive immunity but is also involved in the regulation of NK cell effector functions. Since tumor cells express significant levels of GITRL, and ligation of GITR on NK cells markedly reduces cytokine production and cellular cytotoxicity, our data indicate that GITR-GITRL interactions play an important role in the escape of tumor cells from innate immune surveillance.
Analysis of the Human T Cell Receptor (TCR) Repertoire from Birth to Old Age Suggests That TCRVβfrequencies Are Established Independent of HLA.
Authors: Melenhorst J. Joseph , Zeilah Josette , Sosa Edgardo , Follmann Dean , Hensel Nancy F. , Barrett Austin J.
Published: April 2020

Human T cell development occurs in two waves of development and proliferation: first, early T cells expressing the TCRb chain but not the α-chain are selected for functional TCRβ protein independent of HLA recognition, a process called β-selection; second, thymocytes expressing both the α- and β-TCR are selected for intermediate affinity for self-MHC/ self-peptide complex. This latter process is referred to as positive selection. We sought to determine whether the peripheral TCRVβ frequencies in the naïve T cell repertoire start off at a fixed rank order with minimal skewing as would be expected from a predominantly β-selected repertoire. A total of 22 TCRVβ proteins was quantitated by flow cytometry in a group of 20 unselected umbilical cord blood (UCB) samples (a kind gift from Dr. P. Rubinstein, NY Blood Center, NY), consisting of >80% naïve T cells as defined by CD27+CD45RA+ staining in CD4+ and CD8+ cells. A common rank order of TCRVβ protein frequencies was found in both CD4 and CD8 T cell subsets (figure 1). Median TCRVβ frequencies in CD4 and in CD8 cells of UCB were statistically not significantly different from the frequencies in adult donor CD4 and CD8 cells (Wilcoxon signed rank test; p > 0.2). Furthermore, the percentages of CD4 cells expressing a particular Vβ correlated significantly in CD8 cells (figure 2) with some Vβ proteins being predominantly expressed by either CD4 (Vβ2, Vβ5.1) or CD8 (Vβ14, Vβ7) cells. Our data therefore conform to the prediction that the TCRVβ frequencies are dominantly shaped by β-selection, and not by interactions of the αβTCR/ co-receptor with MHC/ antigen complexes during thymic selection.

Figure 1. TCRBV in UCB CD4+ (top) and CD8+ (bottom) T cells Figure 1. TCRBV in UCB CD4+ (top) and CD8+ (bottom) T cells Figure 2. Comparison of TCRBV protein expression frequencies in CD4 and CD8 cells of UCB Figure 2. Comparison of TCRBV protein expression frequencies in CD4 and CD8 cells of UCB
Generation of CD4+ Regulatory T Cells by Retroviral Transduction of CD4+CD25− T Cells Either with the Full-Length or with a Common Exon-2 Negative Variant of Human Foxp3.
Authors: Mutis Tuna , Verdonck Leo F. , Aarts-Riemens Tineke , Emmelot Maarten
Published: April 2020

The forkhead/winged helix transcription factor, Foxp3 is a key element for the differentiation of CD4+CD25+ regulatory T (Treg) cells. While murine Foxp3 gene is expressed as a single full-length transcript, we observed that transcription of human Foxp3 gene usually reveals two different mRNA products with ~ 100 base pair difference. Sequencing of these products revealed that one transcript represents the full-length Foxp3, while the other transcript appears to be an alternative splicing product that lacks the exon-2. Using a specific primer set that amplifies the region between exon-1 and exon-3 we found that both exon-2pos and exon-2neg variants are preferentially expressed in CD4+CD25+ cells with high levels of expression in CD4+CD25hi cells. Analysis of individual Treg clones generated by limiting dilution of CD4+CD25hi cells revealed that both Foxp3 variants are simultaneously expressed in Treg clones. However, quantitative-real time PCR analyses, performed using a primer set that amplifies only the full-length Foxp3 and another set that amplifies both exon-2neg and exon-2pos variants, indicated that expression levels of the full-length Foxp3 gene do not correlate with expression levels of total Foxp3 in individual Treg clones, in PBMC of healthy donors and in leukemia patients after donor lymphocyte infusions. Remarkably, some donors expressed little or no full-length Foxp3 while expressing considerable amounts of total Foxp3, suggesting a predominant expression of the exon-2neg variant in CD4+CD25+ T cells of these donors. To determine the role of individual Foxp3 isoforms in Treg cell-differentiation, the full-length and the exon-2neg variants of the Foxp3 gene were cloned into retroviral vectors and transduced separately into highly purified CD4+CD25− cells. After a brief in vitro culture, T cells transduced with the Foxp3 isoforms were sorted to high purity by flow cytometry using the GFP marker gene and expanded to high quantities by nonspecific stimulation with allogeneic feeder cells, PHA and IL-2. Phenotypical and functional analyses of expanded cells revealed that T cells transduced with both Foxp3 variants expressed high levels of CD25, intacellular CTLA-4 and CD62L; were anergic to stimulation via CD3 and suppressed the CD3/CD28 triggered proliferation of autologous and allogeneic CD4+CD25− cells. These results reveal that both isoforms of human Foxp3 are functionally intact and can convert CD4+CD25− cells into Treg cells. Current efforts are focused on testing the in vivo capacity of in vitro expanded, Foxp3 transduced T cells to prevent Graft versus Host Disease (GvHD) in a xenogeneic model where GvHD is induced by administration of human T cells in Rag2, gamma chain double knockout mice.
Treatment of Childhood T Cell Lymphoblastic Lymphoma (TLL), Results of the SFOP LMT96.
Authors: Bergeron Christophe , Perol David , Pacquement Helene , Perel Yves , Coze Carole , Gandemer Virginie , Vannier Jean P. , Mechinaud Francois , Schmitt Claudine , Leverger Guy , Baruchel Andre , Patte Catherine
Published: April 2020

After LMT81 and SFOP LMT89 protocols (both modified LSA2L2), the SFOP LMT96 was designed according to BFM 86 back bone (Reiter and al, J Clin Oncol, 13:359.1995). The aim of LMT96 protocol was to increase chemotherapy intensity early (day 8) and late (after the intensification phase). From February 1997 to December 2003, 83 unselected patients (pts) with newly diagnosed TLL were included in LMT96 protocol: classic corticosteroid prephase (d1 to d7); induction phase with Cyclophosphamide(CPM: 1g/m2) and high dose methotrexate (HDMTX: 3g/m2 infusion over 3h) at d8, followed by Vincristine, Daunorubicin (40mg/m2) d15, 22, 29; 8 injections of Asparaginase, 2 intrathecal injection MTX at d8 and d15 and prednisone for 4 weeks; two consolidation phases with HDMTX, Cyclophosphamide (CPM), Cytarabine and Asparaginase; one interphase with HDMTX and; an intensification phase similar to a phase II BFM study, a maintenance phase with Mercaptopurine/MTX including initially six intensified monthly pulse phases alternating either HDMTX/Asparaginase, or Cytarabine/prednisone. The total duration of treatment was 18 months for stage I, II and III and 24 months for stage IV. Cranial radiotherapy (18Gy) was only performed in pts with CNS disease. 66% were boys, median age was 10.5 y [1.9 – 17.2] and 88% had a mediastinal involvement. According to St Jude classification, 5 had a stage I or II (6%); 47 had a stage III (57%); 24 had a stage IV [ (63%), 18 bone marrow (BM) involvement, 4 CSF+ and 2 both]. 7 pts received corticosteroid before diagnosis and were classified as stage X and treated as stage IV. After induction phase and consolidation (d 65), the complete remission rate was 95%. 1 pt had a progressive disease and died and 2 pts in partial remission are alive after salvage treatment with local radiotherapy or intensification. Up to now, 8 pts relapsed between 3.7 months and 18 months (median=10.5 months) and 1 pt who developed a glioblastoma before relapsing at 69.2 months. All relapses were mediastinal, 3 were combined with BM and 1 with BM and CSF. The median of FU is 4 years. Five-year EFS is 87% [CI95%: 79–94] and OS is 89% [CI95%: 82–96]. In conclusion these results are as high as those of BFM experience. The intensified monthly pulse courses for 6 months did not seem decrease the relapses on therapy. Early intensification at d8 (with CPM and HDMTX) is feasible with a BFM regimen. Omission of prophylactic cranial irradiation while using a short duration (3 hours) of HDMTX infusion did not jeopardize the outcome. We suggest that our early intensification might be combined with a classic BFM regimen.
Primary CNS Lymphoma Treated with HD-Methotrexate, HD-Busulfan/Thiotepa, Autologous Stem Cell Transplantation and Response-Adapted Whole-Brain Radiotherapy: Results of the Multicenter OSHO-53 Phase II - Study.
Authors: Montemurro Michael , Kiefer Thomas , Schüler Frank , Ali Haifa-Katrin Al , Wolf Hans-Heinrich , Herbst Regina , Haas Antje , Helke Kirsten , Theilig Anja , Lotze Christian , Hirt Carsten , Niederwieser Dietger , Schwenke Matthias , Krüger William , Dolken Gottfried
Published: April 2020

Purpose:

This multicenter study investigated the efficacy and safety of high-dose methotrexate (HD-MTX) induction followed by high-dose busulfan/thiotepa with autologous stem-cell transplantation (HD-BuTT) and response-adapted whole-brain radiotherapy (WBRT) in patients with newly diagnosed primary CNS lymphoma.

Patients and methods:

23 patients (median age 55 years) were treated in five centres. Patients received HD-MTX (4h-infusion; 8g/m2; >60y: 6g/m2) on d1 and d10 followed by leucapheresis. Then patients were stratified according to their results on neuroimaging: In case of at least a partial response, HD-BuTT consisting of 16mg busulfan / 10mg thiotepa per kg body weight followed by peripheral stem cell transplantation was given. Patients without response to induction or without complete response after high-dose therapy received WBRT (45Gy) as further treatment.

Results:

16 patients received the planned treatment with HD-MTX followed by HD-BuTT. CR / PR rates for these patients were 19 % / 69 % after HD-MTX, 69 % / 13 % after HD-BuTT, 81 % / 6 % after HD-BuTT plus WBRT, respectively. Included the patients with early WBRT due to toxicity (n=2) and non-responders to HD-MTX induction (n=4) the overall response rate for all 23 patients was 83 % (intention-to-treat). Outcome was significantly influenced by the response to MTX-induction. There were three treatment-related deaths. Irradiated patients (n=9) had a high incidence of severe neurotoxicity leading to death in 3 patients. At a median follow-up of 15 months the median EFS and OS for all patients were 17 and 20 months, after HD-BuTT 27 months and “not reached”, respectively. Patients older than 60 years and younger patients have achieved similar outcomes.

Conclusion:

This study showed that HD-methotrexate induction followed by HD-BuTT is a feasible treatment option for newly diagnosed primary CNS lymphoma. Patients achieving CR after HD-BuTT show no signs of clinical neurotoxicity with median survival not reached yet. Time on treatment is 2–3 months only, but the induction treatment needs improvement to be more effective. WBRT in this study was associated with a high incidence of severe neurotoxicity and should therefore be avoided.
International Primary Central Nervous System Lymphoma Collaborative Group (IPCG) Study on Low-Grade Primary Central Nervous System Lymphoma in Immunocompetent Patients.
Authors: Korfel Agnieszka , Jahnke Kristoph , O’Neill Brian P. , Blay Jean-Yves , Abrey Lauren , Martus Peter , Poortmans Philip M.P. , Shenkier Tamara N. , Batchelor Tracy T. , Neuwelt Edward A. , Raizer Jeffrey J. , Pels Hendrik , Herrlinger Ulrich , Thiel Eckhard
Published: April 2020

Low-grade primary central nervous system lymphoma is a very rare subtype of primary central nervous system lymphoma (PCNSL), for which almost no data is currently available. The purpose of this retrospective study was to characterize the clinical presentation, course and outcome of patients with low-grade PCNSL.

Forty patients (18 male, 22 female) from 18 cancer centers in five countries were identified with a median age of 58 (range, 19–78) years and a median Eastern Cooperative Oncology Group (ECOG) performance status of 1 (range, 0–4). The mean time to diagnosis was 14.8 months (range, 0.25–84). Thirty-two patients (80%) had a B-cell and eight a T-cell lymphoma. Thirty-seven patients (92.5%) showed involvement of a cerebral hemisphere or deeper brain structures, while two evidenced only leptomeningeal involvement, and one patient had spinal cord disease. Treatment was performed in 39 patients: chemotherapy and radiotherapy in 15 (38%), radiotherapy alone in 12 (30%), chemotherapy alone in 10 (25%), and tumor resection alone in two. The median progression-free survival (PFS), disease-specific survival (DSS) and overall survival (OAS) were 61.5 (range, 0–204), 130 (range, 1–204), and 79 (range, 1–204) months, respectively. An age ≥60 years was associated with a shorter PFS (P = .009), DSS (P = .015) and OAS (P = .001) in multivariate analysis.

Low-grade PCNSL differ from the high-grade subtype in pathological, clinical and radiological features. In this study, the long-term outcome was better as compared to the results obtained in PCNSL in general with age ≥60 years adversely affecting survival.
Phase II Study of Denileukin Diftitox (Ontak) for Relapsed/Refractory T-Cell Non-Hodgkin’s Lymphoma.
Authors: Dang Nam H. , Pro Barbara , Hagemeister Fredrick , Samaniego Felipe , Jones Dan , Rodriguez Maria , Goy Andre , Romaguera Jorge , Walker Pamela L. , Younes Anas , Neelapu Sattva , Kwak Larry , Fayad Luis
Published: April 2020

Denileukin diftitox (ONTAK) is a fusion protein combining interleukin-2 (IL-2) and the enzymatically active domain of diphtheria toxin that targets tumor cells expressing the IL-2 receptor. We initiated a phase II study at MD Anderson Cancer Center to evaluate its efficacy in relapsed/refractory T-cell lymphoma excluding CTCL. Denileukin diftitox was administered at 18 mcg/kg/day by IV infusion daily for 5 days every three weeks for up to 8 cycles. Corticosteroid was given before every infusion to reduce the incidence and severity of acute hypersensitivity reaction. Twenty-five patients are currently evaluable for response. Median age was 55 years (range 26–80) and mean number of prior treatments was 2 (range 1–6). Tumor CD25 status was determined by flow cytometry and/or immunohistochemistry, with CD25 positivity being defined as 10% or more tumor cells expressing detectable CD25. Of the 13 patients with CD25+ T-cell lymphomas, there were 4 CR (30.8%) and 4 PR (30.8%) for an overall response rate of 61.6%. Of the 10 patients with CD25- tumors, there were 1 CR (10%) and 3 PR (30%) for an overall response rate of 40%. Two patients had a tumor CD25 of undetermined status. The overall response rate was 48% with 5 CR (20%) and 7 PR (28%). The median progression-free survival for responding patients was 6 months (range, 1–24+ months), with 1 patient with CD25+ ALK-1 negative ALCL having an ongoing CR at 24+ months. Of note is the fact that one patient who experienced a CR and another who had a PR went on to receive allogeneic transplantations while in remission induced by denileukin diftitox. Treatment was well tolerated, with the majority of toxicity being grade 1or 2 and transient. Most common toxicities seen were transient transaminase elevation, hypoalbuminemia and edema. Denileukin diftitox has activity in relapsed/refractory T-cell NHL, and is well tolerated at the dosing scheduled tested. While response to denileukin diftitox is seen in both CD25+ and CD25− T-NHL, drug activity in CD25 expressing tumors is particularly striking.
CD4 T-Helper Responses to the ALK Protein in Patients with ALK-Positive ALCL.
Authors: Ait-Tahar Kamel K. , Hatton Chris S.R. , Pulford Karen K.
Published: April 2020

Anaplstic Lymphoma Kinase (ALK)-positive anaplastic large cell lymphoma (ALCL) has a favourable prognostic outlook compared to ALK-negative AlCL, possibly as a result of the immune recognition of the ALK protein. We have previously shown the presence of both a cytotoxic T cell and an antibody response to the ALK protein in patients with ALK-positive ALCL. The aim of our present study was to investigate the presence of a CD4 T-helper (Th) response in patients with ALK-positive ALCL and in control individuals. Using the TEPITOPE web-based predicitive search algorithm, three 24-mer promiscuous peptides were identified from the ALK sequence as being potentially immunogenic in the context of MHC class II. A gamma-interferon (γ-IFN) and IL-4 ELISPOT assay was used to detect a T cell response in the peripheral blood cells from patients with ALK-positive and ALK- negative ALCL, as well as healthy controls after 6–11 days of culture with the three peptides. ALK278–301 and ALK233–256 were shown to be highly immunogenic in the majority of the ALK-positive patients (see Table). ALK411–434 was immunogenic to T cells from only one of the ALK-positive patients (Patient 4). Cells from none of the two ALK-negative ALCL patients or the five healthy donors showed any reactivity to the ALK peptides. No response to the control irrelevant peptide was observed in any of the ALCL patients or healthy donors. With the exception of one ALK-positive ALCL patient (Patient 2), no significant IL-4 response was recorded in any of the patients or controls. All of the ALK-positive patients presented antibodies to the ALK protein at time of diagnosis.These findings further demonstrate the immunogenicity of the ALK protein and are suggestive of a Th1 type of immune response to the protein. Our findings are of potential prognostic value and open up therapeutic options for those ALK-positive patients who do not respond well to chemotherapy.

Summary of the CD4 Th responses to ALK in ALCL patients and healthy donors None PHA (10 μ g/ml) ALK233–256 (10 μM)- (IFN- γ/IL-4) ALK278–301(10 μM)- (IFN- γ/IL-4) ALK411–434 (10 μM)- (IFN- γ/IL-4) Irrelevant peptide (10 μM)- (IFN- γ) Antibody titres to ALK (IgG isotype) ND= Not done. Results are of triplicate cultures ALK+ve patients Patient 1 12 188 56/10 44/18 22/6 8 1/2250 Patient 2 20 240 126/48 78/52 40/26 18 1/2250 Patient 3 14 48 38/ND 24/ND 12/ND 16 1/6750 Patient 4 6 108 64/8 72/8 22/6 10 1/60750 Patient 5 10 48 36/13 26/18 12/12 14 1/6750 Patient 6 15 132 74/ND 58/ND 28/ND 18 1/6750 Patient 7 10 180 34/28 56/32 12/9 12 1/750 ALK-ve patients Patient 8 14 122 12/10 10/6 12/14 22 −ve Patient 9 16 82 14/ND 12/ND 10/ND 24 −ve Healthy Donors Normal 1 22 148 18/14 22/24 26/12 10 −ve Normal 2 12 18 2/4 6/8 12/2 4 −ve Normal 3 10 38 12/10 12/16 9/4 18 −ve Normal 4 9 172 9/ND 10/ND 6/ND 12 −ve Normal 5 4 108 8/12 8/12 2/1 10 −ve
Dasatinib (BMS-354825), a Multi-Targeted Kinase Inhibitor, Inhibits the Kinase Activity of Wild-Type, Juxtamembrane and Activation Loop Mutant Kit Isoforms Associated with Human Malignancies.
Authors: Schittenhelm Marcus M. , Shiraga Sharon , Schroeder Arin , Corbin Amie S. , Griffith Diana , Lee Francis Y. , Deininger Michael W. , Druker Brian J. , Heinrich Michael C.
Published: April 2020

Activating mutations of the activation loop (AL) of KIT are associated with certain human neoplasms, including a subset of patients with AML,systemic mast cell disorders (SM), seminoma, and Gastrointestinal Stromal Tumors (GIST). KIT AL mutations such as D816V that are typically found in AML and SM are resistant to imatinib (IM, IC50 > 5–10 μM). Dasatinib (BMS-354825) is a novel, oral, multi-targeted kinase inhibitor that targets BCR-ABL and SRC. Due to its potent inhibition of these kinases, dasatinib is currently being tested in clinical trials of patients with imatinib resistant/intolerant CML/Ph+ ALL. Based on previous observations of the ability of certain SRC/ABL inhibitors to also inhibit KIT kinase, we hypothesized that dasatinib might inhibit the kinase activity of both WT and mutant KIT isoforms. The inhibitory potential of dasatinib against WT KIT, KIT mutant isoforms and KIT-dependent downstream pathways was evaluated by immunoblotting. In addition, we evaluated the effects of dasatinib on cellular proliferation and induction of apoptosis. Dasatinib potently inhibited WT, juxtamembrane- (JM) and AL-mutant KIT autophosphorylation. Based on the ability of dasatinib to bind to BCR-ABL irrespective of the ATP AL conformation (inactive versus active), dasatinib was expected to be insensitive to KIT AL conformation. In contrast, we found that the IC50 for KIT autophosphorylation varied significantly among the various KIT mutant isoforms: WT KIT, D816Y, V560G (JM mutation) [IC50 1–10 nM] <D816F [IC50 100 nM] <D816V [IC50 200–250 nM]. These results indicate that the conformation of the KIT AL does influence dasatinib potency. Inhibition of KIT kinase activity by dasatinib reduced cellular proliferation and induced apoptosis in mast cell/leukemia cell lines expressing mutant KIT isoforms. In these cell lines, KIT activates downstream pathways important for cell viability and cell survival such as RAS/MAPK, JAK/STAT and PI3K/AKT. Dasatinib potently blocked activation of MAPK1/2 and STAT3. Inhibition of KIT by dasatinib abrogated phosphorylation of AKT S473, but not AKT T308. This partial inhibition of AKT activation was insufficient to inhibit phosphorylation of p70S6K, a kinase downstream of AKT and mTOR. Combining dasatinib with rapamycin, a known mTOR inhibitor, had an additive to synergistic anti-proliferative effect on cells expressing KIT D816V. Our studies suggest that dastatinib may have clinical activity against human neoplasms that are associated with gain-of-function KIT mutations such as AML, SM, seminoma, and GIST. Combining dasatinib with mTOR inhibitors may further increase efficacy against KIT-driven malignancies.