bluuebunny/crossref_metadata_2025 huggingface dataset:
PD-1 Expression Is Markedly Upregulated on Intratumoral CD4+ and CD8+ T Cells in Follicular Lymphoma and Is Associated with T-Cell Exhaustion.
Authors: Nattamai Durgha , Neelapu Sattva S.
Published: October 2019

Follicular lymphoma is one of the most immune-responsive of all human malignancies. However, immunoregulatory mechanisms in the tumor microenvironment may impair the efficacy of immunotherapies such as vaccines and adoptive T-cell therapy. The inhibitory receptor programmed death 1 (PD1), a negative regulator of activated T cells was recently shown to be upregulated on the surface of HIV-specific CD4+ and CD8+ T cells in humans and was associated with impaired T-cell function. Blockade of the immunoregulatory PD-1/PD-ligand 1 (PD-L1) pathway with antibodies against the PD-L1 augmented the function of HIV-specific CD4+ and CD8+ T cells (Day CL et al, Nature, 2006). To investigate the role of PD-1 in lymphoma, we examined PD-1 expression on peripheral blood mononuclear cells (PBMC) and intratumoral T cells in patients with follicular lymphoma prior to therapy. We observed that PD-1 expression is significantly upregulated on peripheral blood and intratumoral CD4+ and CD8+ T cells in patients with follicular lymphoma as compared with normal donor PBMC. Furthermore, PD-1 expression was significantly higher on intratumoral (mean 61%, range 34% to 86%) compared with peripheral blood CD4+ T cells (mean 25%, range 9 to 40%). Likewise, PD-1 expression was significantly higher on intratumoral (mean 44%, range 31% to 69%) compared with peripheral blood CD8+ T cells (mean 16%, range 9 to 31%). PD-1 expression on CD4+ and CD8+ T cells was associated with impaired type 1 cytokine production (IL-2, TNFa, and IFNg) and blockade of the PD-1/PD-ligand pathway with antibodies against PD-1 significantly enhanced T-cell function. These data indicate that the immunoregulatory PD-1/PD-ligand pathway is operative in patients with follicular lymphoma. Moreover, this pathway of reversible T-cell impairment provides a potential target for enhancing the function of exhausted T-cells in follicular lymphoma in combination with other immunomodulatory strategies such as vaccines and adoptive T-cell therapy.
Efficient Lysis of Myeloma Cells by CD16 Positive Effector Cells with Recombinant Single Chain Bispecific Antibodies Targeting HM1.24.
Authors: Peipp Matthias , Ehlert Claudia , Staudinger Matthias , Bruenke Joerg , Fey Georg , Gramatzki Martin , Repp Roland
Published: October 2019

Different therapeutic options are available for the treatment of multiple myeloma patients, but conventional chemotherapy often is not able to completely eradicate the tumor. After high dose chemotherapy, complete remission with only minimal residual disease could be achieved in many patients, but additional targeted strategies may help to eradicate residual cells and improve prognosis. Therapy with monoclonal antibodies is well established in CD20 positive B-cell lymphomas, but in contrast not many suitable target antigens are defined / expressed by multiple myeloma cells. HM1.24 a surface molecule expressed on terminally differentiated B-lineage cells represents a promising candidate antigen that is overexpressed on multiple myeloma cells. Here the development of a recombinant bispecific single chain Fv HM1.24 × CD16 antibody (tandem format) with novel features is presented. The HM1.24×CD16 bispecific antibody was expressed in 293T cells and purified to homogeneity by two-step affinity chromatography. Binding to HM1.24 and CD16 was demonstrated by immunofluorescence staining and flow cytometry with antigen positive and negative cells. The lytic activity of the bispecific HM1.24×CD16 scFv was evaluated in an antibody-dependent cellular cytotoxicity (ADCC) assay with different myeloma cell lines (RPMI 8226, INA-6, U266, JK6L) and primary patient derived cells as targets. Mononuclear cells (MNC), isolated from healthy donors served as effector cells. The bispecific HM1.24×CD16 scFv mediated efficient lysis of all tested cell lines at concentrations as low as 1 nM. In direct comparison to an HM1.24-IgG1 control molecule, the recombinant bispecific antibody demonstrated superior lytic activity at saturating concentrations and showed significant enhanced killing capacity. In conclusion, the recombinant bispecific HM1.24×CD16 retained its antigen specificity and demonstrated efficient lytic activity against patient-derived tumor cell lines and primary material. These results indicate that the bispecific antibody may be promising as a new therapeutic strategy in multiple myeloma.
Efficient Generation of Tumor-Specific, Cytotoxic T Cells by Genetic Transfer of allo-MHC.
Authors: Stronen Erlend , Abrahamsen Ingerid , Johansen Finn-Eirik , Gaudernack Gustav , Lund-Johansen Fridtjof , Olweus Johanna
Published: October 2019

Immune responses to established cancer are difficult to evoke by vaccination of patients due to various mechanisms of tolerance. In contrast, T cells in allogeneic stem cell grafts can contribute to cure of a number of hematological malignancies by the graft-versus-leukemia (GvL) effect. However, unknown T cell specificities causing life-threatening graft-versus-host disease (GvHD) limit the applicability of this treatment. Selection of GvL activity would increase the efficiency, safety and applicability of allogeneic stem cell transplantation. Here, we describe a novel method for overcoming tolerance by generation of allo-restricted, antigen-specific T cells. These cells specifically recognize and kill melanoma tumor cells presenting the melanoma-associated MART-1 in context of the prevalent major histocompatibility complex (MHC) class I molecule HLA-A*0201. HLA-A*0201-negative peripheral blood mononuclear cells were co-cultured with autologous, monocyte-derived dendritic cells transfected with HLA-A*0201 mRNA and subsequently pulsed with the peptide ELAGIGILTV from the melanoma-associated antigen MART-1. Antigen-specific T cells were detected in 8/10 donors on day 12 using peptide-MHC-pentamers (median 0.28% of CD8+ T cells, range 0-4.17%), and in all donors by day 19. When needed, cells were restimulated on day 12 with HLA-A*0201 mRNA-transfected and peptide pulsed autologous EBV-transformed lymphoblastoid cell lines (EBV-LCL). MART-1 pentamer+ T cells from five donors were sorted and expanded. The cytotoxic T cell lines (CTLs) responded with production of interferon-γ, degranulation and lysis of target cells when stimulated with peptide-pulsed EBV-LCL, even at low peptide concentrations (10−5 M). Similar responses were seen to melanoma cell lines expressing HLA-A*0201 and MART-1 (FM-57, Malme3M). In contrast, MART-1+ melanoma cells lacking HLA-A*0201 (Mel202) induced responses only when transfected with HLA-A*0201. Starting out with 0.5×106 pentamer+ T cells, a potential number of 3.0×109 pentamer+ T cells could be reached four weeks later, representing a 7800-fold expansion Our data show that allo-restricted T cells with high specificity for tumor-associated antigens can be rapidly generated, sorted and expanded to clinically applicable numbers.

Figure Figure
Therapeutic Effectiveness of Adaptive T-Cell Transfer for Minimal Residual Mantle Cell Lymphoma Following High Dose Therapy.
Authors: Joshi Shantaram S. , Munger Corey M. , Hegde Ganapati V. , Weisenburger Dennis D. , Vose Julie M.
Published: October 2019

Mantle cell lymphoma (MCL) is the most aggressive B-cell malignancy with an overall survival of less than four years. Although high-dose therapy followed by stem cell transplantation is effective in reducing the tumor burden and increasing the survival, the patients eventually relapse due to residual lymphoma. Therefore effective therapeutic modalities are needed to target residual MCL. In this study we have developed an immune-based approach to treat minimal residual MCL. HLA matched normal donor mononuclear cells were used to generate dendric cells in vitro. Following characterization, the dendritic cells were fused with the MCL cell line Granta 519 cells to create DC/MCL hybrids. The hybrids were then used to stimulate T cells from the same donor to generate MCL-specific cytotoxic T lymphocytes (CTL). The CTLs were tested for their MCL-specific cytotoxic abilities in vitro against MCL target cells and compared to control T cells or DC primed with MCL lysate or DCs transfected with MCL RNA. HLA matched human breast cancer cell line MDA-231 was used as an irrelevant tumor target. The in vitro results showed the highest MCL specific cytotoxic effects of CTLs generated using DC-MCL hybrids for immunostimulation. As a logical next step, in vivo therapeutic affects the MCL-specific CTLs were tested using a preclinical animal model. Four groups of NOD-SCID mice, with a minimum 10 mice per group, were transplanted with Grant-519 human MCL cell line intravenously. MCL bearing mice were treated with combination chemotherapy (CHOP)over three days followed by transplantation of 1 ×106 mononuclear cells. Ten days after, mice were transplanted with 3 ×106 adoptive T cells weekly for four weeks. The results of the in vivo studies showed that, adoptively transferred T cells that had been stimulated with DC/MCL hybrids were able to significantly inhibit tumor growth in the liver (p = 0.005), kidneys (p = 0.001), and lungs (p= 0.001) of mice with minimal residual MCL compared to control tumor bearing animals as determined by analyzing tumor burden in the organs using virtual images and Neuroinformatica software. When combined with CHOP chemotherapy, adoptively transferred T cells were able to significantly (P = 0.001) extend the survival of the mice by significantly further reducing the tumor burden as compared to mice only receiving the chemotherapy plus unmanipulated T cells. Specificity of the CTL mediated response was also confirmed by cytokine profiling in vitro and in vivo. In summary, we have clearly shown that antigen-primed DCs can be used to activate MCL-specific T cells which, when administered adoptively, are effective in treating minimal residual MCL. Thus these studies lay foundation for a PhaseI/II clinical trial.

(This study was supported by the Lymphoma Research Foundation, New York, NY).
High Avidity Leukemia-Associated Antigen-Specific CD8+ T Cells Preferentially Localize to the Bone Marrow in Patients with Myeloid Malignancies.
Authors: Melenhorst J. Jopseph , Scheinberg Phillip , Chattopadhyay Pratip K. , Gostick Emma , Roederer Mario , Hensel Nancy F. , Douek Daniel C. , Barrett A. John , Price David A.
Published: October 2019

Both acute and chronic myeloid leukemias (AML and CML) and myelodysplastic syndromes (MDS) over-express and present self-antigens such as the HLA-A*0201-restricted proteinase 3 (PR1) and Wilm’s tumor-1 (WT1) epitopes, making these leukemia-associated antigens selectively amenable to immunotherapeutic intervention. Here, we examined the antigen avidity properties of circulating and bone marrow-resident CD8+ T cells specific for PR1 and WT1 in patients with AML (n=11), CML (n=10) and MDS (n=3). A total of 19 bone marrow (BM) samples and 27 peripheral blood (PB) samples were studied both prior to and following stem cell transplantation (SCT). Cognate HLA-A*0201 tetramers with identical TCR docking platforms were produced using three distinct monomeric HLA-A*0201 complexes with differential coreceptor binding properties to dissect the avidity of antigen binding directly ex vivo:

“CD8-null” tetramers, which contain a compound D227K/T228A mutation in the a3 domain of the heavy chain that abrogates CD8 binding; wildtype tetramers; and, “CD8-enhanced” tetramers, which contain a Q115E mutation in the a2 domain of the heavy chain that moderately increases CD8 binding.

We have shown previously that CD8-null tetramers engage only high avidity antigen-specific CD8+ T cells; in contrast, CD8-enhanced tetramers can engage populations of antigen-specific CD8+ T cells with low avidities that fall below the threshold for detection with wildtype tetramers. Using these reagents, we developed a polychromatic flow cytometric panel that enabled the simultaneous assessment of phenotype, function and avidity within antigen-specific CD8+ T cell populations. Either PR1- and/or WT1-specific CD8+ T cells were identified in 12/19 BM samples and 6/27 PB samples. Notably, one of the pre-SCT samples contained only low avidity leukemia-associated antigen-specific CD8+ T cells; in contrast, all of the specific populations identified in the post-SCT samples engaged their cognate antigen with high avidity. In 5/7 patients, analysis of paired BM/PB samples revealed the presence of high avidity PR1- and/or WT1-specific CD8+ T cells confined almost exclusively to the BM. Phenotypic analysis demonstrated a mixture of central and effector memory cells in all cases, thereby confirming that these PR1- and WT1-specific CD8+ T cell populations were antigen-experienced. Thus, high avidity CD8+ T cells specific for leukemia-associated antigens are present in vivo and preferentially localize to BM in myeloid malignancies.
Increasing Functional Avidity of T Cell Receptor (TCR)-Redirected T Cells by Removing Defined N-Glycoslyation Sites in the Constant Domain of Introduced TCR Chains.
Authors: Kuball Juergen , Hauptrock Beate , Wolfl Matthias , Voss Ralf Holger , Strong Roland , Theobald Matthias , Greenberg Phil
Published: October 2019

Adoptive transfer of T lymphocytes transduced with a TCR to impart tumor reactivity has been reported as potential strategy to redirect immune responses to target cancer cells. However, the affinities of most TCRs specific for shared tumor antigens that can be isolated are usually low, in part reflecting the nature of the targeted tumor antigens which are self-proteins. Thus strategies that can increase the affinity or functional avidity of TCRs to be used in therapy to transduce T cells might be therapeutically beneficial. However, current strategies for increasing TCR affinity require extensive and usually random mutagenesis followed by screening the many derived mutations, and must be individualized for each TCR. Since glycosylation impacts the flexibility, movement, and interactions of surface molecules, we tested if removing conserved N-glycoslyation sites in the constant regions of TCR a or b chains can increase the functional avidity of T cells transduced with such modified TCR. We observed enhanced functional avidity and improved recognition of tumor cells by CD8 T cells harboring partially N-deglycosylated as compared to wild type (wt) TCR chains. T cells transduced with wt or N-deglycosylated TCR chains bound tetramer equivalently at 4°C, but tetramer-binding was enhanced at 37°C, reflecting predominantly reduced tetramer dissociation, suggesting an energy-dependent conformational change and/or enhanced clustering of N-deglycosylated TCR chains. The enhanced avidity was evident in the presence or absence of the CD8 co-receptor. This strategy was effective with murine and human TCRs specific for different antigens, and thus should be readily translated to TCRs with any specificity. Thus, transfer of partially deglycosylated TCR chains is a generally applicable strategy that can be used to increase functional avidity of TCR-transduced T cells.
Electrocardiographic Screening for Cardiac Iron in Thalassemia Major.
Authors: Detterich Jon A. , Miller Janelle , Coates Thomas D. , Wood John C.
Published: October 2019

Electrocardiographic abnormalities are common in Thalassemia Major patients as well as in animal models of iron overload. The relationship between these ECG abnormalities and the degree of cardiac iron overload has not been well characterized.

Hypothesis: Standard 12-lead ECG identifies preclinical cardiac iron deposition.

Methods: Fifty-four patients with Thalassemia Major underwent MRI quantification of cardiac iron within one month of a standard 12-lead ECG. Cardiac T2* measurements to quantify iron were performed using a validated multiecho gradient-echo sequence performed on a 1.5 Tesla General Electric CVi scanner. The PR, QRS, QT, and QTc durations as well as P-wave, T-wave and QRS axes and heart rates were measured. Normative data was derived from 20 control patients without detectable cardiac iron (T2* > 20 ms) and age-appropriate norms were created for each ECG criteria. Z-scores for the ECG parameters were then calculated for 34 patients having detectable cardiac iron (T2* < 20ms).

Results: ECG parameters varied significantly with age, with correlation coefficients ranging from 0.39 (QTc interval) to 0.66 (PR interval). Cardiac iron was associated with lower heart rates (Z=−0.44, p=0.06), QTc prolongation (Z=0.48, p=0.04) and leftward shift of the P and T wave axes [P-Axis (Z = −1.53, p<0.001); T-Axis (Z= −1.91, p<0.001)]. In total, 31 of 34 pts with cardiac iron had one or more ECG abnormalities (|Z|>2), compared to only 7 of 20 control pts (p<0.0001). Subjective ECG review was consistent with Z-score. The most common abnormalities associated with cardiac iron were non-specific ST-T wave changes (n = 15), sinus bradycardia (n=4), symmetric T-wave inversions (n = 4) and LVH (n = 4). The presence of any of these findings on ECG predicts detectable cardiac iron with a sensitivity and specificity of 68% and 85%. A combined metric of PR prolongation (Z>2), left shift of P or T axis (Z<–2), bradycardia, LVH, ST changes, or symmetric T-wave inversions produced a sensitivity of 94% and specificity of 75% for detectable cardiac iron.

Conclusions: Cardiac iron overload produces characteristic abnormalities in atrial conduction and ventricular repolarization. ECG may be an effective and inexpensive screening modality for cardiac iron overload.

Figure Figure Figure Figure
Long-Term Effects of Combined Chelation Therapy on Glucose Metabolism of Thalassemic Patients.
Authors: Farmaki Kalistheni , Angelopoulos Nicholas , Berdoukas Vasilios , Tsoumari Ioanna , Pappa Christina , Pappagianni Kiki
Published: October 2019

• The incidence of impaired glucose tolerance and diabetes in thalassemiamajor patients varied from 9% to 15%, depending on the age of assessment and intensity (and compliance) of chelation and transfusion. It is uncertain whether early assessment and tailored chelation can prevent diabetes and preserve pancreatic reserve. There are now reports of endocrine improvements with intense chelation in regards to glucose metabolism which are limited to 3 years of follow up. However, whether the pancreas may regenerate or remodel even with reduction in hemosiderosis is unknown and questions have arisen whether these are lasting improvements reflecting an overtime advantage earned by the intensification of chelation. The aim was to determine the long-term effects of combined chelation therapy on glucose metabolism in beta-thalassaemic patients • From January 2001 to October 2003, 42 thalassemic patients (initially treated with deferoxamine DFO monotherapy) were gradually switched to an individually tailored combined regimen with DFO and deferiprone. Glucose metabolism characteristics were evaluated by Oral glucose tolerance test (OGTT). Each patient was assessed separately before initiating combined therapy, and all patients were thereafter yearly reassessed (June 2005, June 2006 and June 2007). Full biochemical data (4 consecutive measurements) were available in 30 patients (3 got pregnant during this period, 5 denied repeated OGTT and 4 patients with severe and sustained hyperglycaemia started antidiabetic drugs after the second assessment in 2006). Insulin sensitivity and beta-cell function were assessed by calculated indices, using the most widely applied insulin sensitivity index - the homeostasis assessment model (ISI HOMA) which is based on fasting glucose and insulin alone. The area under the curve (AUC) was also calculated for estimating integrated response during OGTT for both glucose and insulin. Significant variations over time for each parameter were examined with ANOVA (general linear model) for repeated measurements. Ferritin levels were significantly decreased (P<0.01),AUCglu decreased (P=0.002)and SCHoma increased (P=0.004) as illustrated in Table 1. 70% of patients had abnormal glucose response during the first OGTT (diabetic response, Impaired glucose tolerance or impaired fasting glucose, according to the latest criteria of the American Diabetes Association published in September 2005). In May 2007, only 20% exhibited abnormal glucose response with the same provocative test (P<0.001,non-parametric McNemar test). Our 6-year follow-up demonstrates that use of combined chelation therapy continues to be of benefit in terms of glucose metabolism in thalassemic patients.

Parameters (Mean value) 1st assessment 2005 2006 2007 Ferritin 2960 551 585 629 SCHoma 133.4 215.6 200.6 209.9 ISI Homa 1.101 0.964 1.085 0.886 AUCglucose 18217 16166 16526 15655
The Synthetic Hest Shock Protein (Hsp) 90 Inhibitor EC141 Induces Degradation of the Bcr-Abl p190 Protein and Apoptosis of Philadelphia Chromosome (Ph)-Positive Acute Lymphoblastic Leukemia (ALL) Cells.
Authors: Ferrajoli Alessandra , Wang Yongtao , O’Brien Susan M. , Faderl Stefan H. , Harris David M. , Van Quin , Hallevi Inbal , Liu Zhiming , Koch Patricia E. , Sensintaffar John , Burrows Francis , Kantarjian Hagop M. , Keating Michael J. , Estrov Zeev
Published: October 2019

High dose chemotherapy of Ph+ ALL is rarely curative and clinical responses to protein kinase inhibitors have been transient. Although new regimens combining chemotherapy with Bcr-Abl kinase inhibitors improve survival, the long-term prognosis of patients with Ph+ ALL remains guarded. Thus, novel therapeutic strategies are needed. Hsp90 is a ubiquitous molecular chaperone protein required for the folding, activation and assembly of mediators of signal transduction, cell cycle control, and transcription regulation. The Hsp90 inhibitor EC141 (Biogen Idec, Inc.) blocks the chaperone activity of Hsp90 and induces proteasomal degradation of it’s client proteins. Because Hsp90 is a chaperone of Bcr-Abl we investigated the activity of EC141 against the Ph+ ALL B-cell lines Z-119, Z-181 and Z-33 (Estrov et al. J Cell Physiol166: 618, 1996; Leukemia10:1534, 1996). First we studied the effect of EC141 on Hsp levels in Ph+ ALL cells. EC141 (50 nM) down-regulated the protein levels of Hsp90 and upregulated those of Hsp70. Then, the effect of EC141 on the proliferation of Ph+ ALL cells was evaluated using the MTT assay. EC141 inhibited the growth and metabolic activity of Z-119, Z-181 and Z-33 Ph+ ALL cells in a dose-dependent manner at concentrations ranging from 1 to 100 nM. Similar results were obtained with primary bone marrow cells from patients with Ph+ ALL. Using the ALL blast colony culture assay we found that EC141 inhibited the proliferation of marrow-derived ALL colony-forming cells in a dose-dependent fashion. To explore the mechanism of action Z-181 were incubated cells with increasing concentrations of EC141; immunoprecipitation and Western immunoblotting were used to detect changes in cellular protein levels. EC141 degraded the Bcr-Abl p190 protein and inhibited the phosphorylation of CrkL in a dose-dependent manner. Furthermore, exposure of Z-181 cells to EC141 resulted in a time- and dose-dependent activation of procaspase 3, cleavage of poly (adenosine diphosphate-ribose) polymerase and apoptotic cell death as assessed by Annexin V. Taken together, our data suggest that EC141 degrades the Bcr-Abl p190 protein, inhibits proliferation, and induces apoptosis of Ph+ ALL cells. Additional studies aimed at investigating the in vivo activity of EC141 in Ph+ ALL are warranted.
Oncogenic NK-Like Homeodomain Proteins Inhibit Etoposide-Induced Apoptosis in T-ALL Cell Lines by Activation of the Polycistronic miR-17-92 Transcript.
Authors: Nagel Stefan , Venturini Letizia , Meyer Corinna , Drexler Hans G. , MacLeod Roderick A.F. , Scherr Michaela
Published: October 2019

Homeobox genes of the NK-like familiy, including TLX1, TLX3 and NKX2-5, are ectopically activated in T-cell acute lymphoblastic leukemia (T-ALL) cells mostly via chromosomal aberrations. The pathologic function of these closely related genes is still unclear. Here we analyzed their effect on the C13ORF25 gene, containing the miR-17-92 cluster. Micro RNAs (miRNAs) are a class of small non-coding RNAs which are part of an evolutionarily highly conserved intracellular mechanism, regulating gene expression by hybridization to complementary sequences usually located in the 3′untranslated region of coding transcripts. The primary transcripts (pri-mRNA) are processed to short mature miRNAs, mediating either inhibition of mRNA translation or mRNA cleavage. Aberrant expression of specific miRNAs is involved in oncogenesis as recently described for several human malignancies. The miR-17-92 polycistron encodes miRNAs which decrease E2F1 protein expression. Transcription of both E2F1 and miR-17-92 is induced by MYC, itself a target of E2F1, generating a highly regulated interactive network. Depending on the cellular context, E2F1 performs conflicting tasks by triggering proliferation or inducing apoptosis. We investigated the expression of the miR-17-92 cluster in T-ALL cell lines. Real-time RT-PCR analysis of both miR-17-92 pri-mRNA and mature miRNAs revealed different expression levels in these cells, suggesting a possible implication of the NK-like homeodomain proteins in the regulation of the miR-17-92 cluster in T-ALL. HELA cells transfected with TLX1 or NKX2-5 expression constructs showed elevated miR-17-92 pri-mRNA expression, demonstrating an activating effect. Lentiviral-mediated overexpression of NKX2-5 in the T-ALL cell line MOLT-4 consistently showed increased miR-17-92 pri-mRNA levels and decreased E2F1 protein amounts. For functional analysis of these downstream targets, another T-ALL cell line (PEER) was lentivirally transduced with expression constructs for either miR-17-92 or E2F1, resulting in reduced or elevated E2F1 protein levels, respectively. Overexpression of miR-17-92 or E2F1 did not significantly influenced the cell proliferation. However, induction of apoptosis by treating these cells with etoposide, an inhibitor of topoisomerase II, indicated that overexpression of miR-17-92 and E2F1 resulted in enhanced and reduced cell viability, respectively, as analyzed by MTT assay. In summary, these data indicate an activatory effect of oncogenic NK-like homeodomain proteins on miR-17-92 expression, reducing E2F1 protein levels and thereby enhancing survival of leukemic T-cells.
Frequent Mitochondrial DNA Mutations and Polymorphisms in Long-Term Survivors of Childhood Acute Lymphoblastic Leukemia.
Authors: Lipshultz Steven E. , Walker Vernon E. , Torres Salina M. , Walker Dale M. , Barry Elly , Miller Tracie L. , Scully Rebecca E. , Sallan Stephen E.
Published: October 2019

Progressive myocardial dysfunction, which can lead to cardiomyopathy, congestive heart failure, and sudden cardiac death, is a well-recognized late effect of several agents used in the treatment of childhood cancer. While the mechanism of this damage is not yet fully characterized, mounting data from rodent models implicate treatment-induced mitochondrial DNA (mtDNA) mutations as one potential mechanism of late cardiotoxicity. In the current study, the Dana-Farber Cancer Institute Acute Lymphoblastic Leukemia (ALL) Consortium collected peripheral blood mononuclear cell samples from 93 long-term ALL survivors who were 4 or more years post-doxorubicin and other multi-agent therapy including asparaginase, corticosteroids, vincristine, and methotrexate, as well as radiation therapy. Data from healthy newborn children (n=45) represented a negative control and data from children receiving highly active antiretroviral therapy (HAART) (mean age=11.3 years; SD=2.61; n=51) provided a positive control for mtDNA mutations. Maximum cumulative doxorubicin dose varied among patients by treatment protocol (median=300 mg/m2; range 45 mg/m2 to 470 mg/m2). Median age at treatment was 4.5 years (range=0.5 to 20.8 years) and at mtDNA screening, 14.9 years (range=6.0 to 41.1 years). The mitochondrial tRNA genes and flanking regions were screened via PCR-based denaturing gradient gel electrophoresis (DGGE). Preliminary data showed 47 confirmed polymorphisms or mutations in 39 of 93 ALL survivors screened (42%), occurring in PCR products from 6 of 13 primer sets. The mutation incidence in HIV positive controls receiving HAART was 35 changes in 25 of 51 patients (49%). To date, the negative control population data suggest the occurrence of very few to no polymorphisms or mutations. Most mtDNA changes in ALL survivors were distinct from those in children receiving HAART, and included some sequence variants with suggested pathogenic characteristics. Analysis of cardiac function is ongoing. These data suggest that childhood ALL and its treatment may lead to mutations, over-expression of rare polymorphisms, and the induction of persistent changes that when spontaneously occurring have been associated with clinically significant cardiac effects. Conclusive evidence that doxorubicin-containing multi-agent therapy leads to persistent mtDNA mutations fundamental to cardiac dysfunction might allow for the isolation and prevention of these cardiotoxic effects.
Dynamic In Vivo Imaging Reveals That Leukemia-Induced Changes in the Bone Marrow Microenvironment Alter Mechanisms of Metastasis.
Authors: Colmone Angela , Krishnamoorthy Veena , Sipkins Dorothy A.
Published: October 2019

Tissue microenvironments have been shown to critically regulate cancer cell survival and proliferation. Conversely, while tumor growth can induce neovascularization, the impact of other tumor-induced changes in the microenvironment are less well understood. Here we utilize a xenograft model of Nalm-6 pre-B acute lymphoblastic leukemia (ALL) in SCID mice to examine how tumor growth alters the bone marrow (BM) microenvironment. Using dynamic confocal and multiphoton in vivo imaging, we find that tumor growth dramatically down-regulates expression of the chemokine SDF-1 in the BM microvasculature in areas of leukemic proliferation. SDF-1 has been shown to play a key role in cancer cell metastasis for numerous cell types including Nalm-6, and directs initial Nalm-6 homing to specific SDF-1-positive vascular beds. In contrast, we found that Nalm-6 introduced in mice previously engrafted with leukemia home to SDF-1-negative vasculature, predominantly at the advancing tumor margin. Furthermore, inhibition of chemokine signaling by pertussis toxin pre-treatment of Nalm-6 cells demonstrates that Nalm-6 homing in engrafted mice is chemokine-independent. In summary, these findings suggest that leukemic growth profoundly alters the mechanisms underlying the metastatic process by inducing changes in the host vascular microenvironment. These changes may increase the efficiency of the metastatic process and/or the advancement of the tumor margin. Therapies directed at blocking tumor metastases, therefore, may need to be tailored according to tumor stage.
Genetic Profiling of Adult Acute Lymphoblastic Leukemia Cells by Single Nucleotide Polymorphism Oligonucleotide Microarray.
Authors: Okamoto Ryoko , Ogawa Seishi , Akagi Tadayuki , Kato Motohiro , Sanada Masashi , Miller Carl W. , Kawamata Norihiko , Haferlach Claudia , Haferlach Torsten , Koeffler H. Phillip
Published: October 2019

Acute lymphoblastic leukemia (ALL) is a malignant disease of bone marrow cells, resulting from accumulation of genetic alterations of these cells. We analyzed 74 adult ALL samples by single-nucleotide polymorphism DNA microarray (SNP-Chip) using the new algorithm AsCNAR (allele-specific copy-number analysis using anonymous references). 71 samples (96%) showed genomic abnormalities in a mean 4.5 chromosomes including duplications, deletions and loss of heterozygosity with normal copy number [we call this uniparental disomy (UPD)]. About 25% of samples had a normal karyotype but each had genomic changes detectable by SNP-Chip. Importantly, 21 cases (28%) had UPD, and 29% of these had 9p UPD. Other genomic defects included deletions of p16INK4A in 18 cases (24%), deletions of ETV6 in 7 cases (9%), and hyperdiploidy (>50 chromosomes) in 3 cases (4%). In contrast, we also analyzed 399 pediatric ALL samples and deletions occurred in p16INK4A (28%) and ETV6 (22%) and 29% cases had hyperdiploidy. Hyperdiploidy is associated with a good prognosis and occured much more frequency in pediatric ALL (29%) than adult ALL (4%) which may in part explain the better prognosis in pediatric ALL compared to adult ALL. Also, small copy number changes were detected in adult ALL including deletion of B-cell differentiation genes: EBF (4 cases, 5%), Pax5 (5 cases, 7%) and IKZF (Ikaros) (8 cases, 11%), as well as, deletion of miR-15a and miR-16-1 (2 cases, 3%), which is often found in CLL. Amplification of Rel and BCL11A occurred in one case and amplification of Akt2 occurred in another case. Moreover, we found PAX5/ETV6 fusion in one case (1%); in comparison, 14 of 399 pediatric ALL cases (4%) had PAX5 fusion genes. In summary, we discovered hidden abnormalities including small copy number change and UPD in adult ALL and identified differences between adult and pediatric ALLs. In the future, routine SNP-Chip analysis may provide novel subclassification criteria for ALL and identify unique therapeutic targets.
Therapy Related Acute Myelogenous Leukemia and Myelodysplastic Syndrome in Patients with Acute Lymphoblastic Leukemia Treated with the Hyper-CVAD Regimen.
Authors: Verma Dushyant , Thomas Deborah , O’Brien Susan , Faderl Stefan , Koller Charles , Pierce Sherry , Estey Elihu , Garcia-Manero Guillermo , Cortes Jorge , Kantarjian Hagop , Ravandi Farhad
Published: October 2019

Secondary malignancies including myeloid neoplasms occur infrequently in patients with acute lymphoblastic leukemia (ALL). This has been well documented in the pediatric population but not for the adult patients. We have reviewed retrospectively 641 patients with de novo ALL, treated with the hyper-CVAD regimen or its variants, at M D Anderson Cancer Center from 1992 to 2007. Fourteen patients (2.18%, 7 female, 7 male) developed secondary acute myelogenous leukemia (AML) or myelodysplastic syndrome (MDS). Six patients had AML and 8 MDS. Median age at the time of ALL diagnosis was 52 years (range, 23 – 66). Cytogenetics at ALL diagnosis was diploid in 10, pseudo-diploid with −2 in 1, t(9;22) in 1, and unavailable in 2 patients. Frontline therapy included hyper-CVAD in 7, hyper-CVAD with rituximab in 6, and hyper-CVAD with imatinib in 1 patient. Karyotype at the time of AML/MDS diagnosis was −5, −7 in 8 patients, diploid in 1, miscellaneous in 1, inv(11) in 1, t(4;11) in 1, and unavailable in 2. Secondary AML/MDS developed at a median of 31 months (range, 11 – 74) after the start of ALL therapy. Treatment for the secondary AML and MDS was varied and included cytarabine plus anthracycline based regimens for AML and high risk MDS. One patient with MDS received arsenic trioxide, 1 clofarabine, and 1 decitabine. The response rate to treatment for AML/MDS was complete remission (CR) in 2, partial remission in 6 and no response in 6 patients. Five patients (all MDS) underwent an allogeneic stem cell transplant and all but one died at a median of 3 months (range, 0.5 – 11) after the transplant. One patient is alive and in CR 14 months after a haplo-identical sibling transplant; 2 other patients are alive 3.5 and 4 months after the diagnosis of MDS. The median overall survival after the diagnosis of secondary AML and MDS is 8.3 months (range 3.5+ to 18.5+). We conclude that secondary AML and MDS occur infrequently (2.18%) in adult patients with de novo ALL treated with the hyper-CVAD regimens and response to their subsequent therapy is poor.
Evaluation of Clinical Judgement at Initiation of AML-Therapy in Elderly Patients - A Prospective Randomized Trial.
Authors: Schuler Ulrich S. , Hanel Mathias , Bodenstein Heinrich , Einsele Hermann , Pflueger Karl-Heinz , Schmitz Norbert , Wagner Thomas , Wandt Hannes , Herbst Regina , Kaiser Ulrich , Ehninger Gerhard
Published: October 2019

Treatment decisions for AML in elderly patients are based on a more individualized approach than in younger patients. Increasing age is associated with both decreasing efficacy of conventional therapy, increasing influence of comorbidity and functional impairment on the overall outcome. In a prospective trial we tried to answer two questions. (A) Comparison of DA and I-MA -Induction (B) prospective evaluation of the clinical judgement of the haematologist concerning the overall prognosis of the patient. Physicians requesting the randomization by FAX had to give their impression on a 5 point Likert-scale, whether they regarded the overall prognosis of the patient as inferior, comparable or superior to the respective age group, prior to the first dose of chemotherapy. In part (A) Patients >60 years were randomized between DA (Daunorubicin 45mg/m2 d3−d5 +Ara-C 100mg/m2 d1–d7) and I-MA- induction therapy. In a single arm-study, it had been claimed by others [ASH 2002 abstract 1337] that I-MA (Mitoxantrone 10 mg/m2 d1-3, Ara-C 1 g/m2 2x/d on d1,d3,d5, d7) in patients >60yrs results in high CR-Rates of 73% in de novo and in 59% in secondary AML. Given the expectation of a considerable superiority, several interim analyses were planned, in order to stop the trial early, if I-MA should be shown to improve CR-rates or survival. The most recent analysis is based on 206 patients (more than 60 days on study and evaluable for CR). Concerning part (A) neither CR-rates nor survival differed significantly (overall CR-rate 50%, trend favouring I-MA, survival trend after 1 year favouring DA). In part (B) physicians avoided extremes of the Likert scale. Only in the minority of cases cytogenetic data were available at the time of randomization, but judgement proved to be of value even if this variable was included in the Cox-model. This data show for the first time that clinical judgement (without formal comorbidity scoring or functional assessment) is of value in predicting the prognosis of elderly AML-patients treated uniformly within a randomized trial.

Physician estimate of prognosis Frequency Average Age (median) in years Median Survival (months) p-value (log-rank) inferior 23% 69,7 (69) 4.9 comparable 62.7% 68,7 (69) 7.4 0.124 superior 14.3% 68,5 (68,3) 10.1 0,013
Untreated Patients with Acute Myeloid Leukemia, Age 50–59 Years and Performance Status 2 Should Be as Eligible for Targeted Therapy as Older Patients.
Authors: Tsimberidou Apostolia M. , Kantarjian Hagop M. , Faderl Stefan , Pierce Sherry , Freireich Emil J. , Estey Elihu
Published: October 2019

Background: The early mortality rates in older patients (generally defined as those > 60 years) following standard therapy for acute myeloid leukemia (AML), has spurred numerous trials of “targeted” treatments, typically limited to this age group. Here we examine whether younger patients with Zubrod performance status (PS) 2 might also be candidates for these therapies.

Patients and Methods: We reviewed the records of 1841 patients 40 years or older with newly diagnosed AML (no acute promyelocytic leukemia) who received cytarabine-containing therapy at M. D. Anderson from 1980 to 2007. We chose patients with the following pretreatment characteristics because they are conventional criteria for entry onto trials of new therapies: PS 0–2, bilirubin < 2 mg/dL, and creatinine < 2 mg/dL. We calculated death rates by age and PS at 28 and 42 days after initiation of induction therapy.

Results: Induction death rates in patients with AML by age and performance status The 28-day death rates in patients age 50–59 years with PS 2 were similar to those in patients ≥ 60 years with PS 0–1, and the 42-day death rates in the patients age 50–59 years with PS 2 were similar to those in patients age 60–79 with PS 0–1.

Conclusion: Because of the high death rates following AML therapy in patients age 50–59 with PS 2, these patients should be eligible for targeted treatments now limited to older patients.

Age, yrs Performance Status No. of Patients Dead by day 28, % Dead by day 42 % 40–49 0–1 288 7 9 40–49 2 38 5 5 50–59 0–1 405 4 6 50–59 2 85 14 15 60–69 0–1 423 8 11 60–69 2 111 17 17 70–79 0–1 301 12 16 70–79 2 124 23 33 ≥80 0–1 45 13 20 ≥ 80 2 21 38 43
Mirasol™ Pathogen Reduction of Packed Red Blood Cells (PRBCs) and Apheresis Platelet (Aplt) Concentrates Decreases Neutrophil Priming Activity Generated during Storage.
Authors: Ambruso Daniel R. , Thurman Gail , Marschner Susanne , Goodrich Ray
Published: October 2019

Introduction: During storage of cell containing components including PRBCs and Aplts, biologically active compounds such as lysophosphatidylcholine and other lipids are generated. Identified by their ability to enhance the fMLP stimulated respiratory burst in neutrophils, these biologic response modifiers (BRMs) have been implicated in the pathogenesis of transfusion related acute lung injury (TRALI). The Mirasol pathogen reduction system is a process that uses riboflavin and UV light to modify nucleic acids, reduce infectious pathogen load and inactivate white blood cells in blood components. An additional effect of the Mirasol in PRBCs and Aplts is a decrease in priming activity accumulated during storage.

Methods: PRBCs were produced by standard technique from whole blood donation. PRBCs were treated with Mirasol (8) and stored; untreated, washed and stored in AS-3 additive solution (2); or produced untreated and stored in AS-3 (6). For platelets collected on Trima, one set of products was treated with Mirasol procedure on day 0. Another set was exposed to 25Gy of gamma irradiation. The control group was untreated and stored under standard conditions. Samples were removed on day 0 and 43 (PRBCs) and day 0, 5, 7 (Aplts); cells removed from residual supernatant/plasma by centrifugation and frozen at -70°C. Samples (10% by volume) were assayed to measure the ability to enhance the fMLP stimulated superoxide anion production (O2−) by peripheral blood neutrophils. O2− was measured as SOD inhibitable cytochrome c reduction and expressed as a ratio of the response to plasma followed by fMLP compared to fMLP alone. Priming by platelet activating factor (PAF) was a positive control. Priming is defined as >1.5 fold increase in O2− over the results for fMLP alone.

Results: Samples from Mirasol treated PRBCs show no enhancement of fMLP stimulated O2− production either at day 0 or after 43 days of storage (Top figure). Washed controls exhibit a similar pattern. Unwashed controls demonstrate a small amount of priming on day 0 which further increased on day 43. For Aplts (Bottom figure), day 0 samples exhibited minimal priming. For Mirasol treated products, no changes were seen on days 5 and 7. In contrast, both gamma irradiated and untreated Aplts showed an increase comparable to that seen with PAF.

Conclusion: Treatment of PRBCs and Aplts with Mirasol reduced the generation of priming activity normally developed during storage, possibly representing a decrease in production of BRMs including priming lipids. Mirasol treatment of cell containing components may reduce one class of etiologic agents associated with TRALI and may be a potential approach to reduce risk for this adverse event of transfusion.

Figure Figure
Deficiency of the VWF-Cleaving Protease ADAMTS13 Results in Increased Leukocyte Rolling and Adhesion in Mice.
Authors: Chauhan Anil K. , Kiucka Janka , Brill Alexander , Walsh Meghan T. , Wagner Denisa D.
Published: October 2019

von Willebrand factor (VWF) is synthesized in megakaryocytes and endothelial cells and stored in a-granules and Weibel-Palade bodies, respectively. VWF levels are elevated in both chronic and acute inflammation. ADAMTS13 (A D isintegrin-like A nd M etalloprotease with T hrombo s pondin type I repeats-13) is a metalloprotease that cleaves ultra large von Willebrand factor (ULVWF) multimers quickly after its release from endothelium. Recent studies have found that VWF promotes leukocyte adhesion in vitro and that ADAMTS13 activity is reduced in inflammation and sepsis. We hypothesized that by cleaving ULVWF multimers, ADAMTS13 not only inhibits thrombosis, but also attenuates leukocyte rolling and adhesion. Using intravital microscopy, we found more leukocyte rolling/min on the unstimulated veins in Adamts13-/- mice (Mean ± SE: 98 ± 16) compared to WT (Mean ± SE: 35 ± 6, P<0.001), n=18–20 from 10–11 mice per group. This process was dependent on VWF because the number of leukocytes rolling in Adamts13-/-/Vwf-/- veins was similar to that in Vwf-/-. Significantly increased soluble P-selectin and VWF concentrations were found in the plasma of Adamts13-/- compared to WT mice as quantitated by ELISA. In addition, endothelial P-selectin surface expression was increased in Adamts13-/- mice compared to WT. These results suggest elevated release of Weibel-Palade bodies in Adamts13-/- mice. Notably, circulating platelets were not activated in the absence of ADAMTS13. Upon stimulation of the mesentery with histamine, leukocyte rolling was slower in Adamts13-/- veins compared to WT. Furthermore, upon stimulation with the inflammatory cytokine TNF-alpha (i.v) 3.5 h prior to surgery, the number of leukocytes adhering/250 um was significantly increased in microvenules (diameter of 25–30 um) of Adamts13-/- mice (Mean ± SD: 21 ± 6) compared to WT (Mean ± SD: 12 ± 5, P<0.001), n=10–11 mice per group. This firm adhesion was also dependent on VWF because the number of adherent leukocytes in veins of Adamts13-/-/Vwf-/- was similar to Vwf-/-. Our studies indicate a crucial role for ADAMTS13 in preventing excessive spontaneous Weibel-Palade secretion and in attenuating leukocyte rolling and adhesion to ultra large VWF presented by endothelial cells during inflammation.