bluuebunny/crossref_metadata_2025 huggingface dataset:
Structural Analysis of a BAX-BIM SAHB Complex Reveals a Novel BH3 Interaction Site on BAX for Therapeutic Activation of Apoptosis
Authors: Gavathiotis Evripidis , Suzuki Motoshi , Davis Marguerite L. , Pitter Kenneth , Bird Gregory H. , Katz Samuel G. , Tu Ho-Chou , Kim Hyungjin , Cheng Emily , Tjandra Nico , Walensky Loren D.
Published: April 2020

BAX is a pro-apoptotic protein of the BCL-2 family stationed in the cytosol until activated by a diversity of stress stimuli to induce cell death. Because of its role as a critical gatekeeper of cell death, pharmacologic modulation of BAX has the potential to respectively activate or inhibit apoptosis in hematologic diseases characterized by unrestrained cell survival or pathologic cell death. BAX activation is believed to be a highly regulated, multi-step process involving an interaction-triggered conformational change, mitochondrial translocation, and oligomerization that ultimately leads to mitochondrial dysfunction and apoptosis. Anti-apoptotic proteins, such as BCL-2 and BCL-XL, counteract BAX-mediated apoptosis. Although an interaction site that endows anti-apoptotic proteins with survival functionality has been defined and drugged, an activation site has not been identified for BAX, rendering its explicit trigger mechanism unknown. We previously developed Stabilized Alpha-Helix of BCL-2 domains (SAHBs) that target BCL-2 family proteins in situ, directly initiate BAX-mediated mitochondrial apoptosis in vitro, and suppress leukemia in vivo. Here we demonstrate by NMR analysis that a BIM SAHB peptide binds BAX at an interaction site that is distinct from the canonical binding groove characterized for anti-apoptotic proteins. In five distinct assays that measure ligand-induced BAX activation, BIM SAHB directly and dose-responsively triggers BAX. The specificity of the BAX-BIM SAHB interaction is highlighted by point mutagenesis that abrogates functional activity in vitro and in cells, confirming that BAX activation is initiated at this novel structural location. Thus, we report the first structural analysis of a BH3 death domain alpha-helix bound to a full length pro-apoptotic multi- BH domain protein, unveiling a novel site of protein interaction for direct activation of BAX and pharmacologic modulation of apoptosis.
Presence of Residual Thromboemboli at Least Six Months after a First Episode of Symptomatic Pulmonary Embolism: DO Perfusion Scintigraphy and CT-Angiography Agree?
Authors: Dentali Francesco , Donadini Marco , Cosmi Benilde , Squizzato Alessandro , Bozzato Silvia , Ageno Walter
Published: April 2020

Introduction. The natural history of residual thromboembolic obstructions after pulmonary embolism (PE) and the clinical utility of imaging techniques at follow up are not completely known; in particular, to our knowledge, there are no clinical studies that compared the accuracy of angio-computed tomography (angio-CT) and perfusion scintigraphy (Q scan) in detecting residual thromboemboli after at least 6 months from a first episode of symptomatic PE.

Methods. Consecutive patients on anticoagulant treatment for a first episode of symptomatic PE diagnosed by angio-CT scan and/or Q scan underwent angio-CT and Q scan after 6 to 12 months from the index event. Exclusion criteria were previous PE, recurrent symptomatic PE or deep vein thrombosis (DVT) during the follow-up period. All diagnostic tests were independently assessed by one expert radiologist unaware of the clinical status of the patients. The results of the tests were classified as no signs of recanalization or reperfusion, partial resolution or complete resolution. The agreement beyond chance between tests was assessed by kappa statistics.

Results. Twenty-five patients (14 males, 56%, mean age 59.8) were included after a mean of 7.2 months (217 days, standard deviation 58 days) after acute PE. PE was diagnosed by CT alone in 13 cases, by high probability Q scan alone in 2 cases and by both CT and Q scan in 10 patients. Eleven episodes (44%) were considered idiopathic, 11 (44%) were secondary to transient risk factors, 3 (12%) patients had cancer; concomitant DVT was detected in 17 (68%) patients. At follow-up, Q scan showed no signs of reperfusion in 1 patient (4%), partial resolution in 13 patients (52%) and complete resolution in 11 (44%). Angio-CT showed no signs of recanalization in 0 patient, partial resolution in 17 patients (68%) and complete resolution in 8 (32%). Complete resolution was present in both tests in 1 patient (4%), partial resolution was present in both tests in 6 (24%) patients, with a k agreement between tests < 0.2 (slight).

Conclusions. Partial resolution of thromboemboli after a mean period of 7.2 months from a first episode of PE is present at Q scan or at angio-CT in more than 50% of patients, confirming the results of previous studies. However, the results of our study show for the first time that the agreement between these two tests is low. Thus, caution should be used when interpreting the results of either of these tests to monitor patients with previous PE.
R-FND Followed by Radioimmunotherapy for High-Risk Follicular Lymphoma
Authors: McLaughlin Peter , Neelapu Sattva , Fanale Michelle , Rodriguez Maria , Ayala Ana , Pro Barbara , Hagemeister F.B. , Younes Anas , Neel Susan , Fowler Nathan , Hess Mark , Kwak Larry W.
Published: April 2020

Follicular lymphoma (FL) patients, (pts) with high-risk features using the FL International Prognostic Index (FLIPI) have an expected 5-year survival of only about 50% with conventional therapy. With the incorporation of anti-CD20 monoclonal antibody (mAb) therapy, results are improving (e.g., Buske, Blood2006; 108: 1504). Starting in 2003, we have treated high-risk (FLIPI ≥3) FL pts with R-FND (rituximab, fludarabine, mitoxantrone, dexamethasone) for 4 cycles, followed by radioimmunotherapy (RIT) with ibritumomab tiuxetan, and subsequent rituximab maintenance. Results for the first 35 pts are: complete (CR) and partial (PR) remission 83% and 14%; 3-year overall (OS) and failure-free survival (FFS) 89% and 74% (median follow-up 24 mo.). RIT converted 5 PR pts to CR. Toxicity was mainly hematologic. Five pts did not receive RIT, one because of neutropenia after R-FND. Following RIT, platelet and neutrophil nadirs were 28 and 0.3, occurring at 4–7 weeks. 16 pts required transfusions, and 27 received growth factors. 13 pts had infections, only 2 of which were grade 3. Recovery occurred by 3 weeks in most, with prolonged cytopenias in 6. There has been 1 case of myelodysplasia. In conclusion, the additional complexity of this RIT intensification strategy is warranted in this high-risk FL population, resulting in OS and FFS outcomes that are better than non-mAb therapies, and at least as good as published chemotherapy-rituximab combination therapy.
Long Term Responses to Fludarabine and Rituximab in Waldenstrom’s Macroglobulinemia
Authors: Treon Steven , Branagan Andrew , Ioakimidis Leukothea , Soumerai Jacob , Patterson Christopher , Wasi Parveen , Emmanouilides Christos , Frankel Stanley R. , Lister T. Andrew , Morel Pierre , Matous Jeffrey , Gregory Stephanie A. , Kimby Eva K.
Published: April 2020

Fludarabine and rituximab are commonly used in combination in the treatment of Waldenstrom’s macroglobulinemia (WM), though long term outcome of this regimen remains to be defined. We therefore examined the outcome of 43 WM patients treated on a clinical trial whose eligibility included < 2 prior therapies, and no previous nucleoside analogue or rituximab treatment. Therapy consisted of 6 cycles (25 mg/m2/day for 5 days) of fludarabine and 8 infusions (375 mg/m2/week) of rituximab over 31 weeks. 43 patients were enrolled with a median age of 61, and median prior therapies of 0. Responses were: CR (n=2); VGPR (n=14); PR (n=21); MR (n=4); for an overall and major response rate of 95.3% and 86.0%, respectively. At best response, median bone marrow disease involvement declined from 55% to 5% (p<0.00001), while serum IgM decreased from 3,840 to 443 mg/dL (p<0.00001), and hematocrit rose from 31.2% to 38.0% (p<0.0008). The median time to progression for all patients was 51.2 months, and was longer for untreated versus previously treated patients (77.6 vs. 38.4 months; p=0.017), as well as for those patients who achieved ≥ VGPR versus 88.3 vs. 36.9 months; p=0.049). Grade ≥ 3 toxicities included neutropenia (n=27); thrombocytopenia (n=7); pneumonia (n=6), including two patients who succumbed to non-PCP interstitial pneumonia; peripheral neuropathy (n=2); limbic encephalitis (n=1); hemolytic anemia (n=1). With a median follow-up of 40.3 months, we observed transformation to aggressive lymphoma (n=3); myelodysplasia (n=1); acute myelogenous leukemia (n=2); bladder carcinoma (n=1); and carcinoma of unknown primary (n=1) among 8 patients. The results of this study demonstrate that fludarabine and rituximab is an active regimen in WM, though short and long term toxicities need to be carefully weighed against other available treatment options.
90y Ibritumomab Tiuxetan as Initial Treatment for Follicular Lymphoma (ZEUS Protocol). An Italian Cooperative Study Group
Authors: Carella A. M. , Nati S. , Orcioni G. Fraternali , Petrini M. , Galimberti S. , Vitolo U. , Cascavilla N. , Zinzani P.L.
Published: April 2020

Background: Approximately 80% of patients with Follicular Lymphoma present with disseminated disease, which is consider incurable with standard treatment. Monoclonal antibodies labeled with radionuclides can potentially deliver cytocidal doses of radiation to all sites of disseminated disease. Anti-CD20 radioimmunotherapy has resulted effective in patients who have had relapse after chemotherapy or who have refractory. More recently, radioimmunotherapy (131I-Tositumomab) has been tested also in previously untreated patients with very good results (Kaminsky et al. NEJM, 2005;352:441).

Methods: This study is an open-label, prospective, phase II evaluation of efficacy and safety of a single course of 90Y-ibritumomab tiuxetan at a standard dose of 15 MBq/kg (or 0.4 mCi/kg) in untreated follicular NHL patients. From February 2007, 27 patients with stage III–IV FL received at diagnosis a single course of treatment with 90Y Ibritumomab Tiuxetan (registered as Zevalin). The drug for this study has been suppored by Bayer-Italy. Fifteen patients have enough follow-up for a first evaluation. Eligibility criteria were as follows: CD20 positive follicular lymphoma grade 1 or 2; an age of at least 18 years; no prior therapy, Ann Harbor stage III or IV; at least one bidimensional measurable lesion; involvement of 25% or less of the marrow by lymphoma on trephine biopsy and an absolute neutrophil and platelet count of >1.500×103/ml and 100.000×103/ml, respectively.

Results: Ninety-three percent (14/15 patients) of the patients had any response, and 73% (11/15 patients) had a complete remission. The use of PCR to detect rearrangement of the BCL2 gene was positive at diagnosis in 7/11 remitters patients; six out of these 7 patients developed molecular response and are in continuous complete remission 4–18 months (median, 9 months) after therapy. After a median follow-up of 10 months (range, 4–18 months), 14 patients (93%) are alive, of whom 10 patients in continous complete remission. One remitter patient died of brain stroke; the cause of death was not linked to Zevalin. No patient required hematopoietic growth factors. By week 4, seven patients developed grade 3 thrombocytopenia and 5 of them required platelet transfusions. Until now, no patient developed signs of MDS.

Conclusion: One single dose of 90Y Ibritumomab Tiuxetan as initial treatment can induce clinical and molecular remission in patients with advanced untreated follicular lymphoma; moreover, hematological toxicity was low and quickly reversible.
Efficacy of 90 Y - Ibritumomab Tiuxetan in Relapsed or Refractory Primary Gastric Non Hodgkin Lymphoma
Authors: Vanazzi Anna , Ferrucci Pier Francesco , Grana Chiara Maria , Pruneri Giancarlo , Crosta Cristiano , Pinto Antonello , Chinol Marco , Calabrese Liliana , Paganelli Giovanni , Martinelli Giovanni
Published: April 2020

BACKGROUND: A prospective, single arm, open-label, phase II trial was performed to evaluate efficacy of 90Y-Ibritumomab Tiuxetan at conventional activity of 0,4 mCi/kg in Primary Gastric Non Hodgkin Lymphoma (NHL).

PATIENTS AND METHODS: From May 2004 to May 2008, 13 patients affected by Primary Gastric NHL - either Mucosa Associated Lymphoid Tissue (MALT) or Diffuse Large B Cell (DLBC) NHL, relapsed/refractory disease - were enrolled to receive 90Y-Ibritumomab Tiuxetan at the activity of 0,4 mCi/kg. Median age was 57 ys (range 36–83), 6 female and 7 male. Nine out of 13 patients had gastric MALT NHL, the remaining 4 patients had DLBC NHL. At time of treatment 11 out of 13 patients had stage I/II, 2 patients had stage III/IV disease. Helicobacter Pylori was negative in all patients except one at time of treatment and positive at diagnosis in 5 of them. Median number of previous therapies received was 1 (1–4): all patients except 3 had received prior CT, 7 patients prior Rituximab, no one had received prior RT.

RESULTS: Toxicities were primarily haematological and reversible. All patients had serial testing for HAMA and none developed antibodies to date. MALT-1 gene rearrangement was detected in 3 of the 9 cases analyzed, while none of the cases showed bcl-10 gene rearrangement. All patients are now evaluable for response: 10 out of 13 patients experienced a complete response (CR) (9 MALT, 1 DLBC); progressive disease (PD) was observed in 2 out of 13 patients, both DLBC NHL; finally another patient affected by DLBC NHL did not respond. After a median follow up of 24 months (range 3–50 months) 9 out of 10 CRs are disease free and 5 of them can be considered long term responders at 50, 43, 40, 32 and 31 months, respectively, following radioimmunotherapy (RIT). At the time of the analysis only 1 relapse occurred 17 months after RIT (MALT). Two patients - both DLBC - died due to disease progression. There was no statistically significant associations between response to RIT and the occurrence of the MALT-1 translocation.

CONCLUSIONS: 90Y-Ibritumomab Tiuxetan seems to be very active in patients with primary gastric NHL relapsed or refractory to conventional systemic treatment, especially in gastric MALT NHL. Despite the relative short median follow-up (24 months), we observed durable long-term responses at least in 5 patients. High response rate and durable remissions could be related to the low tumor burden and consequently to higher absorbed doses to tumor, confirming the possible relationship between absorbed doses to tumor, efficacy and duration of response. If preliminary results can be confirmed in larger series, RIT could be offered as valid alternative approach in this setting because of its efficacy and low rate of side effects. These preliminary results warrant further studies.
Antithrombotic Effects of a FXI Antisense Oligonucleotide in Mice
Authors: Zhang Hong , Zhao Chenguang , Crosby Jeff , Mazur Curt , Freier Susan M. , Watt Andy , Siwkowski Andy , Gao Dacao , Black Chris , Monia Brett P.
Published: April 2020

FXI, a component of the intrinsic pathway of coagulation, contributes to the generation of thrombin and is also activated by thrombin on the surface of platelets, resulting in further amplification of the coagulation process. Reports have shown that FXI deficiency in mice or in humans is generally not associated with major bleeding yet is associated with reduced risk for thromboembolic disease. To determine whether targeting FXI with antisense oligonucleotides (ASOs) can produce antithrombotic activity without increasing risk of bleeding, a series of 2nd Generation 2′-O-methoxyethyl modified ASOs were identified that specifically reduce levels of FXI in mice and were evaluated for antithrombotic activity and bleeding potential. Mice were dosed with FXI ASO (ISIS 404071) subcutaneously for three weeks. FXI ASO treatment resulted in a dosedependent and specific reduction in FXI mRNA levels in liver and FXI protein levels in plasma with an ED50 of 3.2 mg/kg. These reductions in FXI levels correlated well with a prolongation of aPTT with no effects on PT prolongation. The anticoagulant effect of FXI ASO treatment also correlated well with antithrombotic activity in a 10% FeCl3 induced IVC thrombosis mouse model across a wide dose range. Furthermore, FXI ASO treatment was generally well tolerated and no prolongation of tail bleeding time was observed at any dose tested, indicating a broad safety margin for FXI ASO targeting. Additional studies using intravital microscopy to study the effects of FXI ASO treatment on thrombosis formation in the mesenteric vein following 10% FeCl3 injury demonstrated that FXI ASO treatment significantly impeded platelet aggregation at the site of vessel injury, thrombus growth and vessel occlusion. Studies to further evaluate the safety of FXI targeting were conducted using a mouse model of intracerebral hemorrhage induced by intracranial injection of collagenase. In this model, FXI ASO treatment had no effect on hemorrhagic size, neurologic deficit score, or mortality at doses well above that needed to produce maximal antithrombotic activity. These results strongly suggest that FXI is an effective and safe target for antithrombotic therapy and that antisense drug technology could be an attractive approach to specifically target FXI for the treatment of thrombotic disorders.
Identification of Hemostatic Modifier Genes Rescuing FVII Deficient Mice
Authors: Rosen Elliot D. , Schroeder Valerie A , Liang Zhong , Suckow Mark
Published: April 2020

FVIItta/− mice are heterozygous for the FVII- null allele and a gene-targeted allele expressing very low levels of FVII. These mice produce very low levels of FVII (<1.0 % of wt levels). FVII+/− and FVIItta/+ mice have each independently been backcrossed into a C57Bl/6 background (thus all the mice in the lineage have had at least 50% of wt FVII levels and there is no selection for compensation for low coagulant activity). The FVII− and FVIItta alleles have similarly been backcrossed extensively into a 129X1/svj background. The FVIItta/− mice were generated in both C57Bl/6 and 129X1/svj backgrounds by crossing FVII+/− and FVIItta/+ animals. In the C57Bl/6 background these low FVII mice suffer intrauterine or perinatal lethality. In contrast FVIItta/− mice generated in a 129X1/svj background survive much longer with 50% surviving more than 6 months. These results indicate there are significant strain differences in the phenotype of mice with a FVIItta/− genotype. To determine the genetic basis of these strain differences we have generated F1 FVIItta/− mice in a 50:50 C57Bl/6 and 129X1/svj background by crossing C57Bl/6J FVII+/tta and 129X1/svj FVII+/− parents. These mice survive long-term similar to FVIItta/− in a pure 129X1/svj background. F1 FVIItta/− males were backcrossed with C57Bl/6J FVII+/− females to generate F1B1 FVIItta/− males. Long-term surviving (>16 weeks) FVIItta/− males were selectively backcrossed with C57Bl/6J FVII+/− females to generate F1B2, F1B3 and then F1B4 FVIItta/− mice. Presumably, the survival of these FVIItta/− mice depends upon the retention of 129X1/svj derived chromosomal regions enabling the survival of low FVII mice. After 4 rounds of backcrossing, the F1B4 FVIItta/− mice retain ~3% of the 129X1/svj genome. DNA from 6 F1B3 FVIItta/− males and all their 81 F1B4 FVIItta/− offspring (total of 87 mice) have been genotyped with a Mouse MD Linkage panel (Illumina) containing 247 informative SNPs distinguishing between 129X1/svj and C57Bl/6J genomes. The initial scan identified 12 chromosomal regions enriched for 129X1/svj derived sequences. To identify putative hemostatic modifier genes, long-term surviving F1B4 low FVII mice have been selectively backcrossed and the DNA of their offspring analyzed with a high density SNP scan in the regions of interest.
Combined Disruptions of the Ribosomal Protein s19 and Pim1 Kinase Genes Are Associated with Increased Myeloid/Erythroid Cellularity and Reduced Apoptosis
Authors: Fröjmark Anne-Sophie , Badhai Jitendra , Schuster Jens , Klar Joakim , Thuvesson Maria , Ellis Steven , Dahl Niklas
Published: April 2020

Diamond Blackfan anemia (DBA) is characterized by reduced proliferation and increased apoptosis of erythroid progenitors. Approximately 25% of patients are heterozygous for mutations in the gene encoding ribosomal protein S19 (RPS19). The molecular mechanisms behind DBA remain unclear and RPS19 was recently shown to interact with PIM1 kinase. To elucidate the phenotypic and cellular effects of this interaction we generated mice strains with the genotypes Rps19 +/− Pim1 +/+; Rps19 +/+ Pim1 −/− and Rps19 +/− Pim1 −/−. We analyzed the mice for peripheral blood and bone marrow parameters as well as apoptotic markers in primary bone marrow cells. The double mutant mice (Rps19 +/− Pim1 −/−) show normal growth and life span with increased white and red blood cell counts when compared to wild type, Rps19 heterozygous, and Pim1 null mice, respectively. Analysis of proteins in bone marrow cells shows that the combination of Rps19 insufficiency and Pim1 deficiency is associated with increased levels of Stat5 as well as the anti-apoptotic markers Mcl-1, Bcl-2 and Bcl-XL. The pro-apoptotic markers Bak, Caspase-3 and p21 show decreased levels whereas p53 remains unchanged. These findings suggest a co-operative effect of Rps19 insufficiency and Pim1 deficiency on cell proliferation in murine hematopoiesis.
Characterization of Novel Natural Mutations in Telomere Binding Protein Factor (TIN2) Identified in Patients with Bone-Marrow Failure Syndromes
Authors: Xin Zhong-Tao , Calado Rodrigo , Savage Sharon A , Lansdorp Peter M , Young Neal S , Ly Hinh
Published: April 2020

We report for the first time potential pathogenic mutations in a known telomeric DNA binding protein factor (TIN2) in patients with acquired aplastic anemia. We examined samples collected from 142 AA patients and 289 healthy controls and found several natural sequence changes in the TINF2 gene, which encodes for the TIN2 protein. Lymphocytes collected from patients with TINF2 variant alleles appeared to grow slower in culture than cells collected from either healthy individuals or those who carried inconsequential polymorphic TINF2 sequence changes (SNPs), consistent with the fact that telomere lengths in these patients were shorter than those of healthy individuals of similar age. We introduced natural mutations in the TIN2 promoter region into a TIN2-promoter driven luciferase reporter plasmid and examined their effect on driving luciferase reporter expression. Using a similar approach, we defined both the regulatory DNA elements and transcriptional factors that control TINF2 gene expression under normal physiological condition. Results obtained from these studies were validated in gel-shift (EMSA) and chromatin immunoprecipitation (ChIP) assays. In addition to mutations in the promoter region, numerous natural mutations were identified in the coding region of the TINF2 gene in patients with AA or Dyskeratosis Congenita (DC). We used co-immunoprecipitation assays to test the effect of natural mutations in disrupting TIN2’s interaction with its known protein partners TRF1, TRF2 and TPP1 of the shelterin complex that protects chromosome ends. The results obtained from these studies will be discussed. To our knowledge, this is the first comprehensive functional analysis of all known mutations in the TINF2 gene in patients with AA or DC. This study offers an insight into how TINF2 mutations can lead to telomere shortening effect and to limited marrow stem-cell reserve and renewal capacity in patients with various forms of bone-marrow failure syndromes.
Age-Adjusted Telomere Length Is Not Associated with Variation in Hematological Parameters in Ageing Women
Authors: Fleury Isabelle , Provost Sylvie , Belisle Claude , Busque Lambert
Published: April 2020

Background. Telomeres play a crucial role in maintaining physical integrity of chromosomes. In the absence of telomerase, telomere length (TL) shortens with each cell division up to a critical threshold where cellular senescence occurs. TL is inversely correlated with age, is longer in women than in men, and demonstrates a strong heritability. Normal blood counts are maintained through out life by an extraordinary number of cell divisions rendering telomere maintenance primordial to prevent stem cell exhaustion. In fact, some cases of bone marrow failure syndromes, such as aplastic anemia and dyskeratosis congenital, have been linked to mutation in the telomerase gene; and stressed hematopoiesis, such at it occurs during the first year following allogeneic bone marrow transplantation induces TL shortening. We hypothesized that individuals with shorter TL may have lower blood counts and a decreased bone marrow reserve. The evaluation of TL as a potential biomarker of ageing hematopoiesis is important in the context of bone marrow transplantation performed with increasingly old donors.

Methods. We measured TL in 1583 women, predominantly aged over 60, all originating from 288 French-Canadian families using a real-time quantitative PCR method that measures the number of telomere repeats relatively to the copy number of a single copy number gene. Telomeres were adjusted for age. Pearson or Spearman correlations were used to determine association between age-adjusted TL (aTL) and hematological parameter according to, respectively, whether or not a normal distribution was observed for these data. A Bonferroni correction was further applied to set the statistical significance threshold.

Results. aTL varied significantly between individuals of the cohort, but no correlation was detected with hemoglobin levels (−0,001; p=0,978), mean corpuscular volume (−0,031; p=0,403); leucocytes (0,055; p=0,139); neutrophils (0,078; p=0,036), monocytes, (0,059; p=0,113), eosinophils (−0,032; p=0,394) and platelets (0,030; p=0,428) counts.

Conclusion. Based on our analysis, TL do not predict blood cells counts in ageing women and may not be a useful biomarker for donor selection. This could also suggest that there is a threshold beyond which TL has an effect on hematopoiesis and that point was not reached in our cohort.
Comparative Genomic Analysis of CLL Genomes Using Affymetrix 50K SNP Versus SNP 6.0 Arrays
Authors: Malek Sami , Wright Whitney , Li Cheng , Shakhan Sajid , Ouillette Peter
Published: April 2020

Unbiased analysis of CLL genomes using intermediate resolution SNP arrays has identified subtypes of del13q14, del17p and del11q as well as high genomic complexity CLL. Additional information on genomic aberrations and polymorphic copy number variants (CNVs) in CLL may be obtained through application of the latest generation ultra-high-density SNP array technology to CLL genome analysis. We have analyzed 50 paired DNA samples (sorted CD19+ cells versus sorted CD3+ cells) from CLL patients with del13q14 type I and del17p on the Affymetrix SNP 6.0 array platform that were previously analyzed on Affy 50K arrays. We have catalogued all somatically acquired lesions as well as all CNVs. In support of data analysis we have refined the software tools PLUT, LOH tool version 2 and dChipSNP.

Results: In this cohort of 50 CLL cases in which we had previously identified a total of 98 subchromosomal losses and gains using the 50K SNP array platform, we have now identified 141 such lesions using the SNP 6.0 arrays. Of these CLL cases, 68% had identical lesion calls, 30% of cases displayed more lesions on the 6.0 arrays than on the 50K arrays and only one case had one lesion not identified using the SNP 6.0 array platform. Thus SNP 6.0 arrays allowed for significantly improved short lesion detection, detection of CNVs and improved measurements of genomic complexity in CLL. The breaks of del13q14 type I and del17p lesions were mapped with unprecedented resolution and in some cases have been localized to genomic regions less than 1 kb in length, thus allowing for PCR-based cloning of breaks. CNVs were identified throughout the genome, and were found in combination with monoallelic, somatically acquired genomic lesions (del17p, del13q14 and others) spanning such CNVs, resulting in patchy biallelic (homozogous) genomic losses within these lesions. Such a mechanism of biallelic gene loss (CNV plus paired, somatically acquired subchromosomal gene loss), occurs in otherwise monoallelic deletions and may contribute to the biology of monoallelic lesions in many cancers. As one example, CNVs were identified in CD3+ DNA from CLL patients that are located at approximately 49.448–49.508 Mb physical position on chromosome 13 that converted to homozygous loss in patients with monoallelic del13q14. These CNVs encompassed parts of DLEU2, TRIM13 and KCNRG but did not include the del13q14 resident miR cluster (miR15a/16-1). Such CNVs within del13q14 may have a role in the biology of CLL as they may result in gene expression changes that mimic somatically acquired del13q14 lesions.
Acute Myeloid Leukemia Induced by MLL-ENL Is Cured by Oncogene Ablation despite Acquisition of Complex Genetic Abnormalities
Authors: Horton Sarah J. , Walf-Vorderwülbecke Vanessa , Chatters Steve J. , Sebire Neil J. , de Boer Jasper , Williams Owen
Published: April 2020

Chromosomal translocations involving the Mixed-Lineage-Leukemia (MLL) gene on chromosome 11q23 are frequent in infant acute leukemia and give rise to the formation of MLL-fusion genes. Several studies have addressed the importance of MLL-fusion activity for the initiation and maintenance of hematopoietic transformation. However, the dependence of established leukemias on MLL-fusion activity has not been previously addressed. We have developed a model for conditional expression of MLL-ENL in hematopoietic progenitor cells, in which expression of the fusion oncogene is turned off by doxycycline. In this study, immortalized myeloid cells conditionally or constitutively expressing the MLL-ENL fusion gene were used to induce acute myeloid leukemia (AML) in vivo. Primary recipients developed AML with a mean latency of 81.4 (±4.8) days. Secondary recipients developed AML with much shorter latencies than primary recipients regardless of whether the leukemic cells were freshly transplanted (26.8 (±6.8) days) or cultured in vitro for one month prior to transplantation (18 (±3.9) days). Genetic analysis revealed that some leukemic cells had acquired gross chromosomal abnormalities such as trisomy 6 or gains and losses of chromosome regions, which were not detected in the immortalised cells from which they were derived. Despite the acquisition of additional genetic abnormalities, the leukemic cells remained dependent upon MLL-ENL expression in vitro and in vivo. The leukemic cells terminally differentiated into neutrophils upon doxycycline treatment in vitro and established leukemias regressed following administration of doxycycline to recipient mice in their drinking water. Leukemic regression was accompanied by the complete loss of leukemic cells from the peripheral blood and differentiation of leukemic cells in the spleen. In 7 out of 34 doxycycline treated mice, remission was not sustained and the leukemias relapsed. However, most of these were shown to have acquired constitutive expression of MLL-ENL. This study demonstrates that leukemic cells are addicted to MLL-ENL expression and suggests that targeting the transcriptional/signalling networks established by MLL-fusion oncogenes in patients with 11q23 rearrangements would be a major therapeutic advance.
Frequently Occurring B-CLL Antibodies Recognize Apoptotic Cells That Expose Non-Muscle Myosin Heavy Chain IIA
Authors: Chu Charles C , Catera Rosa , Didier Sebastien , Zhang Lu , Agagnina Briana , Allen Steven L , Rai Kanti R , Chiorazzi Nicholas
Published: April 2020

B-cell chronic lymphocytic leukemia (B-CLL) is a disease caused by the clonal expansion of a B lymphocyte expressing a unique monoclonal antibody (mAb) of a defined amino acid sequence. At least 27% of B-CLL patients share mAbs with very similar sequences, suggesting common antigen reactivity. A frequently occurring subset of B-CLL patients (at least 49 worldwide) carries an unmutated mAb with a nearly identical or stereotypic sequence that is characterized by a heavy (H) chain encoded by IGHV1-69, IGHD3-16, and IGHJ3 and a light (L) chain generally encoded by IGKV3-20. mAbs from this stereotypic subset (called subset 6) bind a characteristic cytoplasmic protein in human cells. We have recently shown that this protein is non-muscle myosin heavy chain IIA (MYHIIA).

Because MYHIIA is an intracellular protein, we hypothesized that MYHIIA becomes exposed on the cell surface during apoptosis, allowing the mAb of the B-CLL cell to interact with MYHIIA. In this report, we provide evidence that subset 6 mAbs recognize MYHIIA-containing apoptotic cell structures.

Apoptosis of the human T cell line (Jurkat), either spontaneous, or induced by treatment with camptothecin was assessed phenotypically by nuclear DNA rearrangement revealed by immunohistochemical staining with propidium iodide (PI). Costaining with rabbit anti-human MYHIIA followed by fluorescein isothiocyanate (FITC) labeled donkey anti-rabbit IgG revealed that MYHIIA becomes exposed during apoptosis in structures distinct from those containing fragmented nuclear DNA. PI costaining with recombinant subset 6 B-CLL IgG1 mAb and FITC labeled goat anti-human-IgG antibody shows that this B-CLL mAb binds similar apoptotic structures. Finally, Jurkat cells stained with subset 6 B-CLL mAb as before and rabbit anti-human MYHIIA followed by Rhodamine Red labeled donkey anti-rabbit IgG demonstrate that the B-CLL mAb colocalizes with MYHIIA containing structures.

To further study this observation, Jurkat cell apoptosis was assessed by flow cytometry after staining with 7-AAD and Annexin V-Phycoerytherin (AV-PE). Costaining with anti-MYHIIA shows that anti-MYHIIA is exposed in both early (7-AAD+ AV-PE−) and late (7-AAD+ AV-PE+) apoptotic cells, but not in live (7-AAD−, AV-PE−) cells. Similarly, costaining with subset 6 B-CLL mAb demonstrates binding in both early and late apoptotic cells, but not live cells. Intriguingly, not all apoptotic Jurkat cells are MYHIIA+ or B-CLL mAb+.

In summary, subset 6 B-CLL mAbs react with early and late apoptotic cells exposing MYHIIA. Reactivity with a specific apoptotic cell structure(s) may result in the maintenance and expansion of the leukemic clone.
Germline Genes Specific to Chronic Lymphocytic Leukemia (CLL) and Genes Common to CLL, Lymphoplasmacytic Lymphoma/Waldenström’s Macroglobulinemia, and Other Non-Hodgkin Lymphomas Are Important in Susceptibility
Authors: Landgren Ola , Kristinsson Sigurdur Y , Liang Xueying Sharon , Turesson Ingemar , Bjorkholm Magnus , Caporaso Neil E. , McMaster Mary L. , Chanock Stephen , Goldin Lynn R.
Published: April 2020

Background. Evidence from multiply affected families, case-control and population-based registry studies implicate a role for genetic factors in chronic lymphocytic leukemia (CLL). Population-based studies have found CLL and non-Hodgkin lymphomas (NHLs) to aggregate in families. The aim of this study was to expand our understanding on the role of susceptibility genes in CLL and other lymphoproliferative malignancies.

Methods. Using the population-based Swedish Population-, Cancer-, and Multigenerational registries, we identified all CLL patients (n=9,717) diagnosed from 1958–2004 with linkable first-degree relatives (N=26,947). We also obtained information on 38,159 matched controls and their 107,223 first-degree relatives. Cancer risks in relatives of CLL patients were compared with those in relatives of controls using marginal survival models. In parallel, we conducted a candidate gene association study in unrelated familial lymphoma cases (44 CLL, 71 Waldenström’s macroglobulinemia (WM)) from our clinical studies of high risk families (which were selected for having multiple cases of either CLL or WM) compared to 107 spouses from the same families. We tested 1536 single nucleotide polymorphisms (SNPs) in 152 candidate genes in several pathways including apoptosis, DNA repair, immune response, and oxidative stress using a custom Illumina genotyping platform.

Results. In our population-based study, we found relatives of CLL patients to have an increased risk for CLL (relative risk [RR]=8.5, 95%CI=6.1–11.7), lymphoplasmacytic lymphoma (LPL)/WM (RR=4.0, 95%CI=2.0–8.2) and other NHLs (RR=1.7, 95%CI=1.4–2.2), but not for Hodgkin lymphoma, multiple myeloma, or other hematologic tumors. In our SNP-based study, we found several genes (e.g., IL-10, BCL-2, TRAIL, and TRAILR1) to be significantly associated with both familial CLL and WM. In particular, a SNP in the promoter region of IL-10 (rs1800890, −3575T>A), which has been previously detected in other NHL case-control studies, was associated with both CLL and WM.

Conclusions. We conclude that germline genes specific to CLL and genes common to CLL, LPL/WM and other NHLs are important in susceptibility. We found consistent patterns in both our population-based study and our clinical high-risk families. Our findings for BCL-2, TRAIL, and TRAILR1 highlight the important role of apoptosis pathways in the etiology of these lymphomas. Better characterization of early genetic lesions will ultimately provide clues to pathogenesis and allow identification of novel molecular targets.
The Role of CD137 and Its Ligand in Chronic Lymphocytic Leukemia (CLL)
Authors: Baessler Tina , Buechele Corina , Krusch Matthias , Schmiedel Benjamin J , Kanz Lothar , Salih Helmut R
Published: April 2020

Tumor immunosurveillance is dependent on the reciprocal interaction between tumor cells and anti-tumor immunity mediated e.g. by NK cells. This has led to the concept of tumor immunoediting, which incorporates the multitude of mechanisms underlying this dual tumor- and immune-sculpting interaction. Various members of the TNF/TNFR family modulate differentiation, proliferation, activation, and death of both tumor and immune effector cells. Very recently the TNFR family member CD137 has been shown to be induced on NK cells by Fc receptor triggering indicating that not only the Fab region but also the Fc part of a given antibody may be responsible for effects attributed to CD137 modulation (Lin et al., Blood2008, 112: 699). Thus we here studied the role of human CD137 and its cognate counterpart, the CD137 ligand (CD137L) in the interaction of CLL with NK cells. High levels of CD137L expression were detected on B-CLL cells in all investigated patients (n=40). Incubation of CLL cells in the presence of an immobilized CD137-Ig fusionprotein significantly induced the release of the immunoregulatory cytokine TNF demonstrating that CLL-expressed CD137L was capable to transduce bidirectional signals. Furthermore, we found that NK cells of CLL patients displayed substantial CD137 expression. While being absent on resting NK cells, CD137 expression was upregulated on the CD56dimCD16+ but not the CD56brightCD16− NK cell subset of healthy donors upon activation e.g. with IL-2 or IL-15. In addition, CD137 was also induced on NK cells after incubation in supernatants of PBMC of CLL patients. Surprisingly, disruption of CD137-CD137L interaction in cocultures of allogenic NK cells with patient CLL cells by blocking CD137 antibody caused a significant increase in NK cell cytotoxicity. The observed inhibitory effect of CD137L on NK cell reactivity was confirmed in cytotoxicity assays using CD137L-transfectants with mock-transfectants as control. Furthermore, blocking CD137-CD137L interaction also substantially enhanced Rituximab-induced antibody dependent cellular cytotoxicity in an allogenic setting. Importantly, CD137 blockade also substantially enhanced CD107a expression as a surrogate marker for granule mobilization on autologous NK cells within PBMC of B-CLL patients, and this effect was observed both in the absence and more pronounced in the presence of Rituximab. Thus, expression of functional CD137L by CLL cells impairs anti-tumor immunity by diminishing both direct and antibody-dependent cellular cytotoxicity of allogenic and autologous NK cells. Modulation of the CD137-CD137L system might therefore be a suitable therapeutic approach in strategies like antibody therapy which rely on a sufficient NK cell anti-tumor response.
Abnormal Serum Free Light Chain Ratios Are Associated with Earlier Time to First Treatment and Poor Survival in Patients with Chronic Lymphocytic Leukaemia
Authors: Pratt Guy , Mead Graham , Basu Supratik , Jacobs Abe , Holder Roger , Fegan Chris , Pepper Chris , Bradwell Arthur R. , Oscier David , Harding Stephen
Published: April 2020

Introduction: Serum free light chains (sFLC) have prognostic significance in plasma cell disorders. In B-cell chronic lymphocytic leukemia (CLL), a small study found 8/18 (44%) of patients to have abnormal FLC ratios but no assessment of prognostic value was published. The aim of the present study was to determine whether abnormal serum FLC concentrations are indicative of a poor prognosis in CLL patients.

Methods: Sera were analysed from 381 previously diagnosed CLL patients (Stage A 307; B 30; C 26; 18 missing; male: Female Ratio 1.6:1, mean age 71 (29–98)) with samples taken before their first treatment (303) or after treatment (78). The study was approved by the Birmingham Heart of England NHS Trust Review Board. Patients were described using the Binet staging system and measured for prognostic markers including CD38, Zap70, mutational status, β2M and FLC. Kaplan Meier survival curves and Cox proportional hazards regression (age, sex, CD38, Zap 70, mutational status, β2M and sFLC) were calculated using SPSS v14.

Results: 147/381 (39%) patient sera had abnormal sFLC ratios. Kaplan Meier analysis of all deaths showed abnormal ratios were significantly associated with worse survival (n=350, p<0.001). Analysis of deaths attributed to CLL (n=30) also indicated that an abnormal FLC ratio was predictive of shorter survival (p=0.001). However, for deaths not attributed to CLL (n=32), the FLC ratio was not significantly predictive of outcome (p=0.112). For Cox regression analysis (n=228) of deaths attributed to CLL only, three significant, independent, prognostic factors were identified: CD38 (p<0.001), abnormal ratio (p<0.001) and Stage (p=0.027). Analysis of the untreated patient population (n=303), using Kaplan Meier analysis of time to first treatment, found that an abnormal lambda ratio (p=0.04) but not an abnormal kappa ratio (p=0.443) predicted earlier treatment. For patients with an abnormal lambda ratio, the mean time to first treatment was 38 months earlier than those patients with a normal ratio. Cox regression analysis (n=171) of time to first treatment, found 4 significant, independent factors predicting earlier treatment: Zap70 (p<0.001), Age (p<0.001), abnormal sFLC ratio (p=0.001) and Stage (p=0.027).

Conclusions: As shown in other monoclonal gammopathies, abnormal sFLC ratios were associated with poorer outcomes in patients with CLL. Furthermore, in an untreated population, patients with an abnormal lambda sFLC ratio required earlier treatment, indicating a pathological mechanism which is as yet unclear but which warrants further investigation.
Chemoimmuno-Therapy with Fludarabine, Cyclophosphamide and Alemtuzumab in Patients with Relapsed/Refractory CLL: Interim Analysis of the CLL2L Trial of the German CLL Study Group
Authors: Elter Thomas , James Rojin , Stilgenbauer Stephan , Ritgen Matthias , Hallek Michael , Engert Andreas
Published: April 2020

Introduction: Purine nucleoside combination therapy has become the standard treatment approach in B-CLL. In order to enhance efficacy, various combinations with monoclonal antibodies are under investigation. As alemtuzumab has proven to be the most effective antibody in CLL treatment, we developed a multicenter phase II trial combining fludarabine, cyclophosphamide and alemtuzumab in a 4-weekly schedule

Methods: Fludarabine 25mg/m2 iv, cyclophosphamide 200mg/m2 iv and alemtuzumab 30 mg sc were given on days 1 – 3 and repeated on day 29 for up to six cycles. A 2-day escalation of alemtuzumab was administered prior to the first cycle. Minimal residual disease (MRD) was measured by 4-color flow cytometry. Antiinfective prophylaxis consisted of TMP-SMZ DS twice daily 3d/wk and valacyclovir during and at least two months after completion of treatment. CMV antigen in blood was tested in 14-days intervals.

Results: A total of 55 patients of a planned sample size of 61 patients were included in this phase-II study so far, of which 24 patients are evaluable for response and safety analysis. Median age is 63 (range 47–78) years with a medium number of 1 prior regimen; 19/24 patients completed at least four cycles with a median number of 5 cycles. Pretreatment consisted of fludarabine (8/24), fludarabine and cyclophosphamide (13/24) or rituximab combinations (3/24). Thrombocytopenia and neutropenia were the most serious side effects and detailed data will be presented. 3 CMV-reactivations, 1 Herpes-zoster-reactivation and 12 fever of unknown origin have been reported. The overall response rate was 83% with 9 CR(6 CRu) (38%), 9 PR (46%), 3 SD and 1 PD, response was independent of FISH status. A correlation of response with prior treatment was observed, with 100% ORR for fludarabine pre-treatment versus only 77% for those patients pretreated with fludarabine and cyclophosphamide. Updated data of 30 patients and MRD data will be presented.

Conclusions: The concomitant application of fludarabine, cyclophosphamide and alemtuzumab appears as a safe and effective approach for patients with relapsed CLL.
Differential Effects of the BCR-ABL-Inhibitors Imatinib, Nilotinib and Dasatinib on NK Cell Reactivity against Chronic Myeloid Leukemia (CML)
Authors: Krusch Matthias , Salih Julia , Kanz Lothar , Salih Helmut R
Published: April 2020

CML is characterized by the BCR-ABL fusion protein, which mediates the oncogenic signaling. This led to the development of BCR-ABL inhibitors revolutionizing therapy of CML. However, as recently reported for Dasatinib (Schade et al., Blood 111:1366 (2008); Blake et al., Blood 111:4415 (2008)), these agents may impair the activity of immune effector cells like NK cells and T cells. After initiating oncogenic events, development and progression of clinically apparent malignancy is dependent on the evasion of the tumor cells from immunosurveillance. In light of the important role of NK cell reactivity against leukemia we compared the influence of Imatinib, Nilotinib and Dasatinib on the reactivity of both resting and IL-2 activated NK cells against CML cells to identify the compound with the least immuno-compromising side effects. First, the effects of the compounds on NK cell reactivity in concentrations corresponding to plasma peak levels were studied. Dasatinib (200nM) completely abolished NK cell granule mobilization, cytotoxicity and IFN-γ production, while no substantial inhibition was observed with Imatinib (5μM) and Nilotinib (3.6μM) mediated a minor but significant inhibition (p<0.05, Student’s T-test). Presence of the compounds in concentrations corresponding to IC50 levels (Imatinib 600nM, Nilotinib 30nM, Dasatinib 10nM) revealed no influence of Imatinib and Nilotinib, while Dasatinib still significantly reduced NK cell cytotoxicity and IFN-γ production up to 60%. Since Dasatinib, in addition to BCR-ABL, potently inhibits SRC kinases, which are involved in the activation of MAPK pathways and thus crucial for NK cell cytotoxicity, we determined the influence of the compounds on ERK phosphorylation. While no inhibitory effect was observed using Imatinib and Nilotinib, Dasatinib markedly reduced ERK phosphorylation in NK cells. Our data demonstrate that NK cell anti-tumor reactivity is not inhibited by clinically relevant concentrations of Imatinib. While Nilotinib may mediate a minor effect, Dasatinib substantially impairs NK cell reactivity by inhibition of signaling pathways crucial for NK cell effector functions. For a given patient, the choice and dosing of the most suitable BCR-ABL inhibitor may thus require careful consideration of its influence on the immune system, especially in view of the important role of NK cells in the immunesurveillance of residual leukemia.