bluuebunny/crossref_metadata_2025 huggingface dataset:
Hypertension and Obesity in Long Term Survivors of Childhood and Adolescent Cancer.
Authors: Haddy Theresa B. , Mosher Revonda B. , Reaman Gregory H.
Published: April 2020

As a retrospective, single institution study of blood pressure and obesity in survivors of childhood and adolescent cancer, the records of 103 consecutive patients seen in a Long Term Follow Up Clinic from January 1 through June 30, 2004, were examined. Fifty-one patients (49.5%) were female, 52 (50.5%) male. The mean age was 14.6 yr, range 4–33 yr. Ethnic backgrounds were Caucasian 59 (57.3%), AfroAmerican 24 (23.3%), Hispanic 12 (11.6%), Asian 5 (4.9%), and MidEastern 3 (2.9%). Blood pressures for 1–17 yr of age were considered normal if the systolic or diastolic was <90 percentile, prehypertensive if 90–95 percentile, and hypertensive if ≥95 percentile for age, sex, and height. Blood pressures for 18 yr and older were considered prehypertensive if the systolic was 120–139 and/or the diastolic pressure 70–89, and hypertensive if the systolic was ≥140 and/or the diastolic pressure ≥90. Child and adolescent subjects were considered underweight if the BMI was <5 percentile, nonobese if <85 percentile, overweight if 85–97 percenntile, and obese if >97 percentile for age and sex. Adult subjects were considered underweight if the BMI was <18.5, nonobese if 18.5–24.9, overweight if 25–29.9, and obese if ≥30. A total of 29 (28.2%) patients were hypertensive or prehypertensive, and 74 (71.8%) were normal. A total of 38 (36.9%) patients were obese or overweight, 60 (58.3%) normal, and 5 (4.8%) underweight. Most Wilms’ tumor patients, 8 of 10 (80.0%), had hypertension or prehypertension, but only 4 of 10 (40.0%) were obese or overweight. Four of 5 (80.0%) patients with acute myeloid leukemia were obese or overweight, but only 2 of 5 (40.0%) had hypertension or prehypertension. Hispanic (75.0%) and AfroAmerican subjects (66.6%) were likely to be obese or overweight, but only 33.3% of both groups had hypertension or prehypertension. Although the number of study patients is small, our results emphasize the importance of identifying risk factors for hypertension and obesity in childhood and adolescent cancer survivors, particularly in survivors of Wilms’ tumor.
Adverse Events (AEs): Are They Factored into Costs of Hemophilia Care?.
Authors: Aledort Louis M. , Bohn Rhonda
Published: April 2020

Much of the high cost of hemophilia treatment ignores the cost of care for potential AEs related to treatment. Data have been published regarding the thrombotic AEs associated with the use of therapeutic biologics in the management of hemophilia patients with inhibitors. One objective was to quantify the incremental costs of these thrombotic AEs associated with either the available plasma-derived or recombinant products used in managing these patients. Although these occur at low frequency, myocardial infarction, stroke, and pulmonary emboli are reported.

Methods: A review of the literature revealed that no data exist for the cost of these events in hemophilia patients. Using the PharMetrics longitudinal database, it provided us with the frequency and charges associated with thrombotic AEs in hemophilia patients with and without inhibitors. These changes are not inclusive of total replacement product.

Demographic and Clinical Characteristics of the Hemophilia Cohort 1997–2004 Characteristic(Statistic) Hemophilia Patients without Inhibitors (n=5,042) Hemophilia Patients with Inhibitors (n=190) *Myocardial infarction: codes 410.X, 411.X, 412, 429.7 **Pulmonary embolism: codes 415.1X ***Cerebrovascular accident: codes 434.X and 436 Myocardial infarction* 182 patients 26 patients 230 episodes 44 episodes Mean $34,427 $40,110 Median $6,983 $7,070 S.D. $69,680 $64,254 Min-Max $0 – $497,096 $206 – $266,305 Pulmonary Embolism** 78 patients 21 patients Mean 111 episodes 31 episodes Median $18,095 $13,647 S.D. $6,069 $3,186 Min-Max $30,537 $24,738 $4 – $235,638 $63 – $106,082 Cerebrovascular accident*** 128 patients 26 patients Mean 201 episodes 44 episodes Median $27,528 $53,890 S.D. $6,069 $12,554 Min-Max $52,164 $103,017 $0 – 304,714 $32 – $567,055

Thrombotic complications of inhibitor therapy have been recognized for many years. Essentially, little data exist for the costs of these events and none for potential requirements for rehabilitation, loss of employment, and impact on the family. Data from clinical trials may well be a future key factor in determining reimbursement for therapeutics. There is a growing need for longitudinal data to assess the long-term safety as well as the associated costs of potential AEs. Knowledge of these costs will aid treaters in making therapeutic decisions. Coupled with the incidence of these events, incremental costs will aid health care planners to better understand the costs of care associated with hemophilia patients with inhibitors.
Mesenchymal Stem Cells Regulate the Cell Division Symmetry of Hematopoietic Stem Cells by an Integrin Dependent Mechanism.
Authors: Gottschling Sandra , Saffrich Rainer , Seckinger Anja , Krause Ulf , Miesala Katrin , Horsch Kerstin , Wuchter Patrick , Ho Anthony D.
Published: April 2020

Asymmetric cell division is the basal assumption for the generation of a hematpoietic system. On the basis of the murine stromal cell line AFT024 we could demonstrate that cell division symmetry is governed by intercellular contacts between hematopoietic stem cells (HSC) and niche cells. In the adult bone marrow the niche is primarily represented by osteoblasts, whereas during fetal hematopoiesis and pathological extramedullary hematopoiesis in the adulthood mesenchymal stem cells (MSC) are thought to hold this function. Nevertheless data demonstrating an alteration of cell division symmetry by MSC as well as an determination of the type of cellular interaction regulating this process are lacking so far. Therefore the cell division symmetry of individual human PKH-26 labelled CD34+/133+ HSC in contact and non-contact cultures with MSC was monitored for 7d by means of time lapse fluorescence microscopy. For comparison, cultures without feeder layer were monitored. The experiments were performed both, in the absence and presence of early acting cytokines (LIF, IL-6, MIP-1α, G-CSF, TPO, SCF, Flt-3L) promoting the maintenance of primitive progenitors. We found that the vitality and number of dividing cells was exclusively dependent on soluble factors with the highest vitality and number of dividing cells in cytokine augmented MSC cultures (89.9 ± 6.4% vital cells and 98.8 ± 2.7% dividing cells) and the lowest in cultures with neither stromal nor cytokine support (67.9 ± 0.7% vital cells and 41.8 ± 2.5% dividing cells). Nevertheless the percentage of asymmetrically dividing cells was twice as much in MSC contact cultures (22.7 ± 0.7%) than in all other culture conditions (9.1 ± 3.4 to 12.2 ± 2.3%). This demonstrates for the first time that MSC can alter cell division symmetry by a contact dependent mechanism. To identify the type of cellular interactions between HSC and MSC regulating the cell division symmetry, we monitored the latter in prescence of a function blocking β1-integrin antibody that reduced the percentage of asymmetrically dividing cells in MSC contact cultures from 22.7 ± 0.7% to 8.9 ± 4.2%. This was accompanied by a reduction of adherent HSC from 31.6 ± 6.1% to 9.7 ± 4.1% and a loss of functionally primitive cells as determined by a LTC-IC assay. Taken together these data demonstrate for the first time that MSC can alter the cell division symmetry of HSC by a mechanism that is partially dependent on β1-integrin mediated cell contacts.
Reconstitution of Regulatory T Cells after Selective Depletion of CD25+ Host-Reactive Donor Lymphocytes from Allografts and Association with Acute Graft-Versus-Host-Disease.
Authors: Mielke Stephan , Rezvani Katayoun , Solomon Scott R. , Kilical Yasemin , Savani Bipin N. , Wish Laura , Carter Charles S. , Schindler John , Hensel Nancy , Kurlander Roger , Read Elizabeth J. , Vitetta Ellen S. , Barrett A. John
Published: April 2020

Ex-vivo selective depletion (SD) of alloreactive donor lymphocytes by anti-CD25 immunotoxin is a novel method to reduce the severity of graft-versus-host-disease (GVHD) in elderly patients undergoing matched-sibling transplantation (Blood 2005, 106 (3), 1123–9). Nevertheless, there is concern that the concurrent removal of CD4+CD25+ regulatory T cells (Tregs) with the alloactivated CD25-expressing T cells could precipitate more GVHD if depletion of alloreacting cells were incomplete. We, therefore, studied the recovery of Tregs in 11 patients within the first 150 days after SD transplantation by surface phenotyping for CD4 and CD25 and real time PCR for forkhead protein3 (foxp3) gene, shown to be specific for characterisation of Tregs. At transplant patients received a median of 1.0 (0.5–1.5) x108/kg selectively depleted (CD25−) CD3+ T cells and a stem cell product containing 0.25 x104/kg (0.1–5.0) residual unselected CD3+ T cells. Three patients received SD-DLIs on day +60 or day +100. Low average levels of foxp3 gene expression appeared around day +30 post-transplant and remained almost stable until day +150 (Figure A). GVHD occurred in 7 patients (2 grade I and 5 grade II). Acute grade I-II GVHD was restricted to 7 patients with significantly lower foxp3 gene expression at day +30 (for one patient the earliest available sample was from day +60) (Figure B). In contrast to the relatively steady state of foxp3 gene expression average CD4+CD25+ cell numbers were highest during the first 60 days declining thereafter and suggesting the presence of relatively more effector cells in the early post-transplantation period. Chimerism analysis on selected CD4+CD25+ cells showed them to be predominantly of donor origin. These results suggest that foxp3+ Tregs delivered with the stem cell product may undergo modest expansion post-transplant. The absence of high grade aGVHD in these 11 patients probably relates to the efficient removal of host-alloreactive T cells by the SD process. Nevertheless, the occurrence of low-grade aGVHD in patients with the lowest foxp3 gene expression indicates that Tregs are also involved in GVHD protection after SD transplants. As the SD approach is at a very early clinical stage of development this possibility remains to be explored in further studies.

Figure Figure
Expression of Activation Marker, CMRF-44, on Blood CD11c+ Dendritic Cells Is Closely Associated with the Severity of Acute GVHD after Allogeneic Haematopoietic Stem Cell Transplant.
Authors: Lau Jenny , Sartor Mary , Vuckovic Slavica , Munster David , Hart Derek N. , Bradstock Kenneth F.
Published: April 2020

AIM: Dendritic cells (DC) are considered central to the development of graft versus host disease (GVHD) following allogeneic haematopoietic stem cell transplantation (alloHSCT). Both donor and host DC are thought to initiate allogeneic immune responses by presenting host antigens to donor T lymphocytes. We studied the potential of CMRF-44, a novel monoclonal antibody identifying activated circulating blood DC, as a predictive marker of acute GVHD.

METHODS: In a prospective study, peripheral blood was taken from 40 patients twice weekly up to 100 days post-alloHSCT. Circulating myeloid (CD11chi) and plasmacytoid (CD123hi) DCs were enumerated and the expression of CMRF-44 was assessed on CD11chi DC by four colour flow cytometry. Multivariate analyses were performed using a non-parametric Mann-Whitney U-test and Receiver Operating Characteristic (ROC) curves.

RESULTS: Following alloHSCT, the severity of acute GVHD correlated with the number of total DC in the blood (p=0.035). Furthermore, low myeloid and plasmacytoid DC numbers were significantly associated with grade 2-4 acute GVHD (p=0.046 and 0.017 respectively). In 40 alloHSCT patients, 27 developed acute GVHD. CMRF-44 was expressed on CD11c+ DC in all cases prior to the onset of acute GVHD. Of the 13 patients without GVHD, 8 had no circulating CMRF-44+ CD11c+ DC. CMRF-44 expression was independent of the reconstitution of myeloid DC (p=0.73). Patients who had CMRF-44+ CD11c+ DC in more than 20% of their post-transplant monitoring samples were more likely to develop acute GVHD (p=0.001, OR=37.1). In addition, patients with more severe grade 2–4 GVHD had significantly higher percentages of CMRF-44+ CD11c+ DCs (p=0.001). CMRF-44 expression at greater than or equal to 12% of CD11chi DCs had a sensitivity of 87.5 for prediction of grade 2–4 acute GVHD, and a specificity of 91.7.

CONCLUSION: CMRF-44 expression on blood CD11c+ DC is highly associated with the onset of acute GVHD. These results suggest that CMFR-44 may be used as a predictive tool to identify patients at risk of severe GVHD and to direct therapy. Furthermore, it reinforces the potential application of suitably engineered CMRF-44 antibodies for the prevention or treatment of graft versus host disease.
Risk and Clinical Implications of Transformation of Follicular Lymphoma (FL) to Diffuse Large Cell B-Cell Lymphoma (tFL).
Authors: Montoto Silvia , Davies Andrew J. , Matthews Janet , Calaminici Maria , Norton Andrew J. , Amess John , Vinnicombe Sarah , Rohatiner Ama Z.S. , Lister Andrew
Published: April 2020

Between 1971 and 2000, 330 consecutive adults (176M/154F) with newly diagnosed FL were referred to St Bartholomew’s Hospital. The median age was 55 (range 23–90) years; 50 Stage I, 47 Stage II, 56 Stage III, 177 Stage IV. The FLIPI score was ‘low’: 38%, ‘intermediate’: 27%, ‘high’: 35%, with 111 missing observations (LDH or number of nodal sites). 100 patients are still alive, 230 having died between 6 months and 30 years, the causes of death being: disease 106, treatment-related complications 37, other malignancy 30, cardiac disease 20, ‘other’ 18, unknown 19. With expectant management if appropriate and intervention with irradiation, chemotherapy or immunotherapy at times of progression, the response rate was 84% at 1st treatment, declining to 43% at fifth, with duration of remission declining correspondingly. With a strict policy of documenting histology at each progression, a repeat biopsy rate of 70% was achieved. tFL has occurred in 89/330 cases between diagnosis and 16 years, in 25/330 (8%) whilst being managed expectantly or during first therapy and at a relatively constant rate at 2nd, 3rd, 4th and 5th progression, (17%, 16%, 15%, 14% respectively). To date, no patients have transformed after 16 years, with 41 patients ‘at risk’ between 16 and 35 years (Figure 1), 18 in continuous first remission and 23 having had a recurrence of FL. On univariate analysis, risk of transformation is higher with high FLIPI score (p= .009) and advanced stage (p= .02) at presentation. Survival of patients is shorter in whom transformation occurs (p= .0003). Median survival from transformation was 1.3 years, and correlated with ‘stage’ (p= .008) and LDH (p= .03) at the time of transformation, but not with time from diagnosis to transformation. These observations highlight the significance of transformation of FL to t-FL, demonstrating its major clinical importance. The actuarial prediction is that transformation will occur in at least 50% of patients overall with a very poor prognosis for the majority. The ‘time to transformation curve’ supports the hypothesis that there may be two distinct populations of patients who develop FL, one in which transformation is programmed to occur and one in which it is not.

Figure 1. Time to trasnformation. Figure 1. Time to trasnformation.
Signatures of Response to the Proteasome Inhibitor Bortezomib in Diffuse Large B-Cell Lymphoma.
Authors: Feuerhake Friedrich , Monti Stefano , Blank Jonathan , Wu Erxi , Chen Wen , Golub Todd R. , Shipp Margaret A.
Published: April 2020

The proteasome inhibitor, bortezomib (VELCADE®, formerly PS341), has significant anti-tumor activity in several lymphoid malignancies. Reported targets of this broad-based inhibitor include the NF K B pathway (I K B A). Recently defined subtypes of large B-cell lymphoma (LBCL) exhibit constitutive activation of NF K B, prompting us to analyze the efficacy of bortezomib in a panel of 10 DLBCL cell lines. Six of the diffuse LBCL cell lines were sensitive to bortezomib treatment at doses below 10 nM (range IC50 = 2.9 to 6.9 nM) whereas 4 cell lines were resistant at 10 nM (IC50 = 14.8 to 70.2 nM). Baseline proteasomal function, as defined by cleavage of the 20S proteasome-specific fluorogenic peptide LLVY-AMC, was similar in sensitive and resistant DLBCLs; however, the IC50 for bortezomib proteasomal inhibition was somewhat lower in sensitive vs. resistant lines (sens. vs res., p = .04, one-sided t test). Baseline NF K B activity varied widely in the DLBCL cell lines and did not differ in cell lines that were sensitive vs. resistant to bortezomib. Ten nM bortezomib did not inhibit NF K B activity in resistant DLBCL cell lines whereas the same dose reduced NF K B activity in sensitive DLBCL cell lines (sens. vs. res., p < .005, rank test [Mann-Whitney]). However, 5 of 6 sensitive DLBCL cell lines had very low baseline NF K B levels (< 0.5 relative absorbance units) suggesting that NF K B inhibition was not a major factor in bortezomib response and prompting further analysis of additional bortezomib targets. Three sensitive and 1 resistant DLBCL cell line were selected for detailed analyses of transcriptional profiles following bortezomib treatment. We developed an algorithm for identifying genes that were significantly up- or down-regulated in the bortezomib-sensitive cell lines but unchanged in the resistant line. In addition, we utilized gene set enrichment analysis (GSEA) and gene ontogeny (GO) termed enrichment to interpret the molecular signatures of response. Genes down-regulated in response to bortezomib included critical B-cell transcription factors, components of the B-cell receptor signaling cascade and genes regulating mitosis and cell cycle control; up-regulated genes included heat shock proteins (HSP) and multiple proteasomal components. Consistent with the functional data, down-regulation of NF K B target genes was not a common feature in all bortezomib-sensitive cell lines. In contrast, target genes of the c-MYC transcription factor were significantly down-regulated and c-MYC activity was decreased in sensitive (but not resistant) DLBCL cell lines following bortezomib treatment (sens. vs. res., p < .005, rank test). Taken together, the results provide insights into likely mechanisms of action of bortezomib in DLBCL, highlighting c-MYC as a potentially important target and identifying HSP as a complementary target to overcome bortezomib resistance.
Targeting β2-Microglobulin for Induction of Tumor Apoptosis in Human Hematological Malignancies.
Authors: Yi Qing , Yang Jing , Qian Jianfei , Wang Siqing , Wezeman Michele , Kwak Larry W.
Published: April 2020

We discovered that monoclonal antibodies (mAbs) specific to human β2-microglobulin (β2M) induce programmed death of myeloma and other hematological tumor cells. The mAbs exhibited potent in vitro tumoricidal activity on all 14 β2M/HLA-ABC-bearing myeloma, lymphoma, and leukemia cell lines and primary myeloma cells isolated from eight patients. Cell death occurred rapidly, without the need for exogenous immunological effector mechanisms. Although the expression of β2M on normal hematopoietic cells is a potential safety concern, the mAbs seem to be selective to tumor-transformed cells and did not induce apoptosis of normal cells, including T and B lymphocytes and CD34+ bone marrow stem cells. Furthermore, the mAbs were able to selectively kill myeloma cells without damaging normal stromal cells in their cocultures. After binding to cell surface, the mAbs mediated internalization and down-modulation of surface β2M and HLA-ABC molecules. The mAbs induced cell death via inhibiting PI3K/Akt and ERK, activating JNK, upregulating Bad and Bax protein expression, inducing phosphorylation of Bcl-2, and decreasing phosphorylation of Bad, all of which compromised mitochondrial integrity, leading to cytochrome c release into cytosol and activation of the caspase-9-dependent cascade. Inhibitors to pan-caspases or caspase-9, but not to caspase-8, or to JNK, and knockdown of surface β2M and HLA-ABC expression by β2M-specific siRNA, but not control siRNA, abrogated apoptosis of tumor cells induced by the mAbs. Furthermore, anti-β2M mAbs were also active and therapeutic in vivo; After administration subcutaneous or intraperitoneal, the mAbs substantially reduced tumor burdens and retarded tumor growth in SCID mice subcutaneously implanted with human myeloma cell lines ARP-1 or MM.1S. Therefore, such mAbs offer the potential for a novel therapeutic approach to hematological malignancies and possibly to other cancers that express surface β2M. This study also suggests that differential expression of β2M/MHC class I molecules by normal and cancer cells might be a crucial point in the sensitivity and selectivity of anti-β2M mAb-induced apoptosis.
Targeting p62ZIP in Marrow Stromal Cells Is Highly Effective at Inhibiting Myeloma Cell Growth and Osteoclast Formation.
Authors: Kurihara Noriyoshi , Honjo Tadashi , Windle Jolene J. , Shin J. , Roodman G. D.
Published: April 2020

Marrow stromal cells play a critical role in osteolytic bone destruction in multiple myeloma, and promote tumor growth. In particular, adhesive interactions between myeloma cells and marrow stromal cells increase RANK ligand (RANKL), a potent inducer of osteoclast formation, IL-6 and TNF-α production by marrow stromal cells. IL-6 enhances the growth and prevents apoptosis of myeloma cells, and TNF increases production of RANKL and IL-6. Recently, a new member of NF-κB signaling pathway, p62ZIP, has been identified. p62ZIP plays a critical role in NF-κB activation induced by TNF-α and RANKL and is involved in multiple signaling pathways that result in enhanced IL-6 production, tumor cell survival and bone destruction. It is our hypothesis that inhibiting p62ZIP expression will profoundly diminish myeloma growth by blocking the effects of IL-6 produced in the tumor-bone microenvironment in response to TNF-α. Therefore, we used p62ZIP−/− mice to determine the effects of deleting p62ZIP in stromal cells on the growth of myeloma cells. Marrow cells from p62ZIP −/− or wild type mice were used to establish long-term Dexter-type marrow cultures to isolate marrow stromal cells. Marrow stromal cells from p62ZIP −/− or wild type mice were cocultured for 48 h with a GFP-labeled human MM.1S myeloma cell line (MM.1S) and murine and human RANKL, IL-6 and TNF-α levels were determined in conditioned media from these cocultures using commercial ELISA assays. Cocultures of MM.1S with wild type marrow stromal cells resulted in much greater upregulation of murine IL-6 than p62−/− stromal cell coculture (IL-6 in p62−/− stromal cell cultures; 114+70 vs. WT 1900+9 pg/ml). In addition, deleting p62ZIP in stromal cells markedly decreased the growth of tumor cells. Coculture with wild type stromal cells induce 1.4-fold greater increase in MM.1S cell growth at 72 h compared to p62−/− stromal cells. Further, addition of neutralizing antibodies to TNF-α and IL-6 to the cocultures of MM.1S cells with WT stromal cells similarly affected the growth of the MM.1S. Since TNF-alpha can increase the expression of adhesion molecules on stromal cells and tumor cells, we measured expression levels of ICAM-1 and VCAM-1 by Western blot. VCAM-1 and ICAM-1 levels on p62ZIP−/− bone marrow stromal cells were not changed compared to WT stromal cells. We then determined the capacity of p62 −/− cells to support OCL formation by normal spleen CFU-blast. OCL formation was decreased about 50 % in cocultures containing p62−/− stromal cells treated with PTH-rp, IL-6 and TNF-α compare with WT stromal cell culture. These results show that p62 plays an important role in myeloma cell growth and OCL formation induced by cytokines that are upregulated in the marrow microenvironment in patients with myeloma.
GRN163L, a Novel and Potent Telomerase Inhibitor, Inhibits Myeloma Cell Growth In Vitro and In Vivo.
Authors: Shammas Masood A. , Koley Hemanta , Tassone Pierfrancesco , Neri Paola , Protopopov Alexei , Gryaznov Sergei , Chin Allison , Bertheau Robert C. , Mitsiades Constantine , Anderson Kenneth C. , Munshi Nikhil C.
Published: April 2020

Telomerase activity is either low or completely absent in most normal somatic cells; while it is elevated in most cancer cells providing unlimited proliferative potential by preventing telomere shortening. The inhibitors of telomerase, therefore, induce telomere shortening leading to apoptotic cell death in tumor cells while having little or no effect on normal diploid cells. We have evaluated the in vitro and in vivo efficacy of thio-phosphoramidate oligonucleotide specifically targeting the RNA component of telomerase (GRN163L) with demonstrated nuclear uptake by >99% cells without the transfection enhancer. Delivery of GRN163L (1 μM) to MM cells (INA6 and ARP) was specifically associated with complete loss of telomerase activity as early as 6 hrs following exposure and was accompanied by a reduction in myeloma cell growth and survival. Treatment of INA6 cells with GRN163L for three weeks induced 96±4% and 100% cell death at 0.5 and 1 μM concentrations, respectively. ARP cells, which express higher levels of telomerase activity and have longer telomeres, showed 67±4% cell death at 5 weeks with 0.5 μM inhibitor and 82±3% and 100% cell death at 4 and 5 weeks, respectively, with 2 μM GRN163L. The apoptotic cell death was confirmed in 51% INA6 cells at two weeks and in >80% ARP cells at four weeks. Apoptosis was associated with reduction in mean Telomere Fluorescence Intensity (TFI) on interphase chromosomes from 87.1±6.2 in control oligo treated INA6 cells to 36.2±2 (2.4 fold) in GRN163L treated cells. Moreover, GRN163L treatment was also associated with a similar reduction in number of chromosomes with detectable telomeres, indicating development of telomere-free ends. We have confirmed in vivo efficacy of GRN163L in a SCID-hu murine model of multiple myeloma. Following growth of GFP-transduced myeloma cells in the fetal bone chip introduced into the mice, GRN163L was injected on alternate days. In two independent experiments significant reduction in tumor cell growth, as measured by reduction in human myeloma related protein, and better survival than mice injected with control oligo was observed. We have now evaluated efficacy of combination of GRN163L with other novel agents. We have observed synergistic activity with Hsp90 inhibitor 17AAG on myeloma cell death. Addition of 17AAG (0.05 μM) to myeloma cells pre-treated with GRN163L (1 μM) for one week led to complete growth arrest within four days compared to continued growth of cells not pre-treated with GRN-163. These data provide the preclinical rationale for clinical evaluation of GRN163L in myeloma and in combination with Hsp90 inhibitor.
Protein Palmitoylation in Platelet Function: Role in G Protein-Mediated Platelet Activation and Platelet Recruitment into Thrombi.
Authors: Flaumenhaft Robert , Dilks James R. , Sim Derek S.
Published: April 2020

Protein palmitoylation represents the covalent linkage of a 16-carbon saturated fatty acid to a protein. This reversible post-translational modification directs protein-protein interactions as well as protein association with membranes and lipid rafts. Protein palmitoylation participates in ligand-induced signal transduction in several nucleated cells. Its role in platelet activation, however, has not previously been evaluated. We have found that platelets contain the palmitoyl transfer proteins GODZ and HIP14 as well as the palmitoyltransferase, acyl-protein thioesterase 1 (APT1). Thus, platelets possess the basic machinery for regulated palmitoylation. Studies using [3H]-labeled platelets identified several platelet proteins that were palmitoylated following exposure to the protease-activated receptor 1 (PAR-1) ligand, SFLLRN. To determine whether protein palmitoylation functions in activation-induced platelet functions, we infused recombinant APT1 into permeabilized platelets prior to activation with SFLLRN. Infusion of APT1 inhibited platelet protein palmitoylation and completely blocked platelet α-granule secretion induced by SFLLRN. Similarly, the protein palmitoylation inhibitor cerulenin blocked SFLLRN-induced platelet protein palmitoylation, α-granule secretion, and platelet aggregation in intact platelets. To assess the mechanism by which protein palmitoylation affects platelet function, we evaluated the effect of inhibitors of protein palmitoylation on G protein activity. Gαq is essential to PAR-1-mediated platelet activation and is palmitoylated in an activation-dependent manner in nucleated cells. Immunoprecipitation of Gαq from [3H]-labeled platelets showed that it is palmitoylated following activation of platelets with SFLLRN. Both APT1 and cerulenin inhibited SFLLRN-induced palmitoylation of Gαq. In addition, APT1 and cerulenin inhibited SFLLRN-induced GTPase activity as detected using [γ-32P]GTP-labeled platelet lysates. These results show that palmitoylation of Gαq participates in PAR-1-mediated signal transduction. We next used intravital microscopy to determine if protein palmitoylation functions in thrombus formation in vivo. For these experiments, platelets from a donor mouse were incubated with cerulenin and labeled with calcein-AM (green) or incubated with vehicle alone and labeled with calcein-AM red-orange (red). Equal numbers of green and red labeled platelets were then infused into a recipient mouse. The accumulation of cerulenin- and vehicle-treated platelets into thrombi following laser-induced injury of the mouse cremaster muscle was quantified using high speed, digital videomicroscopy. Incubation of platelets with cerulenin resulted in an approximately 50% reduction in their ability to accumulate into thrombi. These studies show that platelet protein palmitoylation is required for thrombus formation as well as for normal platelet function.
Outcomes of Older Patients with Myeloid Leukemias Treated with Myeloablative Intravenous Busulfan-Based Conditioning Regimens and Allogeneic Blood or Marrow Transplantation.
Authors: de Lima Marcos , Couriel Daniel , Ghosh Shubhra , Jones Roy B. , Shpall Elizabeth J. , Giralt Sergio , Hosing Chitra , Kebriaei Partow , Champlin Richard , Andersson Borje S.
Published: April 2020

Older age (e.g. > 55–60 yrs) is a commonly cited indication for reduced-intensity conditioning regimens (RIC) in the treatment of myeloid leukemias. These diseases, however, may benefit from the highest possible dose intensity that can be safely delivered. Here we analyze our experience treating patients (pts) older than 55 yrs. with intravenous (IV) busulfan (Bu)-based preparative regimens.

Methods: Fifty-six pts older than age 55 yrs. with MDS (n=13), AML (n=33), or CML/myeloproliferative disorder (n=10) were transplanted from 6/1997 to 12/2004 on two consecutive protocols. Conditioning were IV fludarabine (Flu) 40 mg/m2 and IV Bu 130 mg/m2 over 3 hr (days -6 till -3) given once daily (BuFlu; n=36), and IV Bu 0.8 mg/kg over 2 hr every 6 hr for 16 doses (days -7 till -4), followed by Cyclophosphamide (Cy) 60 mg/kg on days -3 and -2 (IV BuCy2; n=20). Pts with an unrelated (UD) or mismatched donor received ATG. Graft-versus-host disease (GVHD) prophylaxis was based on tacrolimus and mini-methotrexate.

Results: Median age was 58.4 yrs (55–66.8); 14 pts were older than 60 yrs. Most pts were not in complete remission (CR) at BMT (n=38, 68%, with similar distribution in pts older and younger than age 60). Donors were HLA-compatible siblings (SIBS) in 59% (n=33) and UD in 41% (n=23). UD were used in 5 pts >59 yrs (36%) and in 18 < 60 yrs (43%). Stem cell source was bone marrow (n=23) or peripheral blood (n=33). All but one pt had documentation of donor cell engraftment. Median follow-up of surviving pts (June 2005) was 15 mos (range, 4–49; n=23). Grade II-IV acute (a) and chronic (c) GVHD rates were as follows (pts <60 vs >59 yrs): 38% vs 64% and 45% vs 54%, respectively. 100-day treatment-related mortality (TRM) was 11% (due to graft rejection (n=1), toxicity (n=2); pulmonary hemorrhage with fungal pneumonia (n=1) and aGVHD (n=2). All early 100-day TRM pts were < 60 yrs. Overall, 17 pts relapsed and 33 pts have died. One-year TRM: 29%, n=16 (due to GVHD, n=7, infections, n=4; rejection, n=1; unknown causes, n=1; toxicity, n=2, bleeding, n=1). Only 2 pts > 59 yrs are alive, while the others died of cGVHD (n=5) and disease relapse (n=7).

Conclusion: In this older population of predominantly relapsed pts with myeloid leukemias, major cause of treatment failure was not the preparative regimen employed, but GVHD and disease relapse. An association between intensity of the regimen, age and GVHD cannot be ruled out in this analysis. These results would suggest that age per se should not be used as the main reason for selecting a reduced intensity regimen in patients up to at least age 60 with myeloid leukemias.

Figure Figure
Interaction of MLL Amino Terminal Sequences with Menin Is Required for Transformation.
Authors: Hess Jay L. , Yang Zhaohai , Wang Haoren , Chen Ya-Xiong , Milne Thomas A. , Martin Mary Ellen , Slany Robert K. , Hua Xianxin
Published: April 2020

Rearrangements of the mixed lineage leukemia gene MLL are associated with aggressive lymphoid and myeloid leukemias. The resulting MLL fusion proteins enforce high-level expression of HOX genes including HOX A7 and HOX A9 and the HOX cofactor MEIS1, which is pivotal for leukemogenesis. The mechanism by which this occurs and the relationship to normal MLL function is unknown. MLL and MLL fusion proteins bind with a similar distribution in hematopoietic cells at both promoters and coding sequences of target genes. Our studies suggest that a major mechanism of regulating MLL, which is expressed throughout hematopoiesis, is through modulating it’s binding to target promoters. MLL binds directly to the promoters and coding regions of HOX A7, HOX A9, and MEIS1 only in myeloblasts and not in neutrophils, indicating MLL is physically associated with genes only when they are actively transcribed. Expression of A cluster HOX loci and MEIS1 remains persistently elevated when MLL-ENL or dimerized MLL fusion proteins are expressed. Expression of either fusion protein is associated with increased binding of wild type MLL accompanied by increases in histone acetylation and histone H3 lysine 4, marks that are normally almost completely erased during myeloid differentiation. In addition MLL-ENL induces increased lysine 79 methylation. Both MLL and MLL fusion proteins interact with the tumor suppressor menin via sequences in the extreme amino terminus of MLL. In addition both proteins physically interact with RNA polymerase II, which shows abnormal pausing in the coding regions of HOX genes in Mll null cells. Genetic ablation of menin or expression of a dominant negative inhibitor of the MLL-menin interaction inhibits the growth of MLL fusion protein transformed cells. These findings suggest MLL fusion proteins act in concert with menin, MLL and other coactivators to deregulate HOX gene expression pivotal for transformation.
c-Myb Is an Essential Downstream Target for Homeobox Mediated Transformation of Hematopoietic Cells.
Authors: Slany Robert K. , Bittner Claudia B. , Zeisig Deniz T. , Bach Christian , Hess Jay L.
Published: April 2020

Overexpression of Hox genes is frequently associated with leukemogenic transformation of hematopoietic cells. In particular the abdominal-type HoxA genes together with their homeodomain containing dimerization partners of the TALE (three amino acid loop extension) family are well documented oncogenes in various hematologic malignancies. Paradigmatic examples are Hoxa9 and Meis1 that are coactivated by retroviral insertions in the BXH2 mouse leukemia model. In addition Hoxa9 is a fusion partner of NUP98 in acute myeloid leukemias with a t(7;11) and Meis1 as well as Hoxa9 are critical targets of MLL (mixed lineage leukemia) fusion proteins. Despite their important role in leukemogenesis little is known how they exert their transforming function. Here we used a novel system of transient transformation by an inducible oncoprotein to generate populations of primary hematopoietic cells that overexpress Hoxa9 in combination with Meis1. The RNA inventory of these cells was compared to control cells by Affymetrix array analysis. Interestingly the RNA of c-Myb (myeloblastosis leukemia virus oncogene) and of a known c-myb downstream target (Gstm1) was amongst the top ten genes upregulated by Hoxa9/Meis1. c-Myb overexpression was verified by Northern blot and quantitative RT-PCR. c-Myb levels also responded to the introduction of MLL-ENL, an oncoprotein that upregulates Hoxa9 and Meis1. Small interfering RNAs directed against c-Myb suppressed transformation by MLL-ENL but had no effect on transformation of hematopoietic precursors by E2A-HLF an oncogene that does not work by increasing Hoxa9/Meis1 levels. The anti c-Myb siRNA effect was abrogated by coexpression of a c-Myb derivative with a mutated siRNA target site. The coexpression of a dominant negative c-Myb mutant had a similar but weaker effect on MLL-ENL mediated transformation. Ectopic expression of c-Myb in hematopoietic precursor cells induced a mild differentiation block. However, c-Myb alone was not able to give a positive readout in a serial replating assay under conditions where the combined overexpression of Hoxa9 together with Meis1 was clearly transforming. In summary the results suggest that c-Myb is essential but not sufficient for the oncogenic activity of Hoxa9/Meis1.
Minimal Residual Disease Monitoring by Four Color Flow Cytometry before Second Induction Is an Independent Prognostic Factor in Childhood Acute Myeloid Leukemia - AML-BFM MRD Study Group.
Authors: Langebrake Claudia , Dworzak Michael , Hrusak Ondrej , Mejstrikova Ester , Griesinger Frank , Zimmermann Martin , Creutzig Ursula , Reinhardt Dirk
Published: April 2020

Monitoring of minimal residual disease (MRD) by flow cytometry in childhood acute myeloid leukemia (AML) is still a topic of discussion in terms of the optimal time of analysis and the best antibody combinations. Here we report the largest prospective international series of MRD monitoring (395 samples) in children with AML (n=124).

All children have been treated according to the AML-BFM 98 study. Based upon a CD33/CD34 backbone, a wide panel of antibodies, independent from the initial immunophenotype, was applied at up to four defined time points during treatment (MRD1: day 15, MRD2: before 2nd induction, MRD3: before 3rd block, MRD4 before 4th block). For each of the 12 leukemia associated immunophenotypes (LAIP) and time points, a threshold level based upon a previous retrospective analysis of 149 other children with AML as well as analysis of normal bone marrow was determined.

Regarding all four time points there is a statistically significant difference in the 3-year event free survival (EFS) in those children presenting with MRD positivity at ≥ 3 time points (pEFS 68% vs. 33%, p=0.01). The MRD level at time point 2 has turned out to have the most significant predictive value for 3-year EFS: 66% (n=104) vs. 35% (n=17), plogrank=0.006. Regarding the standard risk group (defined by FAB subtype [M1, M2± Auer rods, M3, M4eo], favorable cytogenetics and morphologically determined treatment response at day 15) only, it is possible to identify a subgroup with poor outcome (20% [n=5] vs. 88% [n=40], plogrank <0.0001). In accordance with this, a multivariate analysis proved the MRD2 as a significant prognostic factor (pChi2=0.023) independent from the known risk factors like FAB subtype, cytogenetics or morphological blast percentage at day 15. As a conclusion, (A) MRD monitoring before second induction has the same predictve value as examining MRD level at four different time points during intensive chemotherapy and (B) the immunologic analysis of MRD before second induction is particulary suitable for risk group stratification and therapeutic consequences in addition to the known risk factors.
Molecular Switch between Tissue Factor-Mediated Signaling and Coagulation.
Authors: Ahamed Jasimuddin , Vesrteeg Henri H. , Kerver Marjolein , Hogg Philip J. , Mueller Barbara M. , Ruf Wolfram
Published: April 2020

Tissue factor (TF) forms a catalytic complex with coagulation factor VIIa and thus triggers coagulation as well as cell signaling through protease activated receptor (PAR) 2. The molecular switch between these two functions remains elusive. We demonstrate in growth arrested keratinocytes that TF’s coagulant function remains unchanged when TF expression levels and TF-VIIa signaling are concordantly downregulated. Thus, coagulation and signaling are mediated by two distinct and differentially regulated pools of TF. TF-VIIa signaling, but not coagulation, is blocked by bacitracin, an inhibitor of protein disulfide isomerase, and TF co-immunoprecipitates protein disulfide isomerases. To test the role of isomerization, we expressed single thiol containing mutants of the cross-stranded, surface exposed TF Cys186-Cys209 disulfide bond. Retaining Cys186, but not Cys209, preserves signaling properties with high affinity for VIIa, but eliminates the TF-dependent coagulation. Cys186-containing TF is surface expressed predominantly in a dimeric form, indicating a trapped isomerization intermediate. Signaling by wild-type, but not the Cys186 dimer, is inhibited by blocking free thiols indicating that thiol-isomerization plays an essential role in regulating TF signaling. Furthermore, we identified a monoclonal antibody that blocks TF-VIIa signaling of wild-type, but not Cys186 dimer TF, suggesting interference with the dynamics of TF isomerization. This human TF specific antibody does not interfere with coagulation, but inhibits the growth of human xenograft tumors in mice. Thus, TF-VIIa signaling, not coagulation, is the primary pathway for TF-driven tumorigenesis. These experiments resolve the molecular mechanism that switches TF-VIIa from coagulation to signaling and show feasibility to block signaling TF for the therapeutic intervention in cancer.
Altered Oncogenic Fitness of Imatinib- and Dasatinib-Resistant BCR-ABL Mutants Is Due to Differential Intrinsic Kinase Activity and Signaling Pathway Selection Defined by Phosphoproteome Profiling.
Authors: Skaggs Brian J. , Burgess Michael R. , Gorre Mercedes E. , Graeber Thomas G. , Sawyers Charles L.
Published: April 2020

Point mutations in the kinase domain of BCR-ABL are the most common cause of resistance to imatinib therapy in CML. E255K and T315I are detectable before the initiation of imatinib therapy in a small population of CML patients, suggesting that certain imatinib-resistant BCR-ABL mutants display altered biological fitness relative to wild-type p210 BCR-ABL. To test this hypothesis, we performed primary murine bone marrow transformation assays using BCR-ABL-expressing retroviruses. In a survey of six clinically imatinib-resistant mutations (Y253F, Y253H, E255K, E255V, T315I, M351T) as well as five additional mutations that confer in vitro resistance to the dual SRC/ABL inhibitor dasatinib (L248R, V299L, T315A, F317L, F317V), we identified several that altered oncogenic fitness either positively or negatively relative to p210. To elucidate the molecular basis for altered transformation potential, we examined the kinase activity of p210 versus mutant full-length BCR-ABL. Enzyme kinetics studies with an optimized Abl peptide substrate as well as gel-based in vitro kinase assays illustrated that intrinsic kinase activity does explain some, but not all, of the mutant oncogenic potency phenotypes. The imatinib- and dasatinib-resistant mutant T315I, for instance, was a gain-of-fitness mutant in the bone marrow assay but exhibited a three-fold lower Vmax than p210. In contrast, T315A, a dasatinib-resistant mutation at the T315 gatekeeper residue, exhibits reduced transforming potential relative to wt p210 and is completely unable to phosphorylate Abl substrates in the kinase assays, although it does retain the ability to autophosphorylate. Subsequent kinase assays on immunoprecipitations from stably expressing IL-3-independent Ba/F3 cells illustrated that certain mutants bind and phosphorylate distinct substrates from p210. We further delineated signaling differences between p210 and the mutant panel by utilizing a mass spectrometry-based global phosphorylation profiling technique. Trypsinized proteins from stably expressing p210 and mutant Ba/F3 cells were immunoprecipitated using anti-phosphotyrosine antibodies, then identified and quantified by two dimensional mass spectrometry. Phosphorylation of specific substrates correlates with gain- or loss-of fitness observed in the bone marrow assay. We conclude that certain BCR-ABL kinase domain mutations, in addition to conferring drug resistance, also affect the oncogenic potency of BCR-ABL by altering intrinsic kinase activity as well as downstream targets. Elucidation of alternative signaling pathways activated by these mutants by the combination of biochemical and mass spectrometric methods may provide novel therapeutic targets for imatinib- and dasatinib-resistant CML.
Donor Lymphocyte Transfusion (DLT) in the Treatment of Relapsed CML after Allogeneic PBSCT - A Retrospective Analysis of 346 Patients by the EBMT Chronic Leukemia Working Party.
Authors: Schmid Christoph , Biezen Anja v. , Porkka Kimmo , Chalandon Yves , Urbano Alvaro , Olavarria Eduardo , de Witte Theo , Hertenstein Bernd , Archese Wiliam , Clark Richard , Kolb Hans-Jochem , Niederwieser Dietger , Guglielmi Cesare
Published: April 2020

DLT has become the gold standard for the treatment of reccurrent CML after allogeneic stem cell transplantation. However, so far, published data are based on DLT following bone marrow transplantation (BMT). During recent years, mobilized peripheral blood stem cell transplantation (PBSCT) have been increasingly performed. To evaluate the efficacy of DLT following PBSCT, we retrospectively analysed the results of 86 patients treated with DLT for CML relapse after PBSCT from HLA-identical family donors. Results were compared to 260 patients receiving DLT for relapse after related BMT. Reduced intensity transplants were excluded.

BMT and PBSCT groups were balanced in terms of patient age (median: 40 vs 42 y), conditioning, stage at transplant (84% vs 76% in first chronic phase (CP) p=.2), and stage at relapse (molecular/cytogenetic: 59% both, CP: 28% vs 20%, advanced: 13% vs 21%, p=.2). However, BMT patients had been transplanted in earlier years (median: 1994 vs.1998, p<.001), had a longer remission after transplantation (median: 677 vs. 275 d, p<.001), and had experienced more aGvHD after transplantation (58% vs. 34%, p<.001). Complete molecular or cytogentic remission was achieved in 71% of the patients, with a trend towards better response in BMT recipients (p=.06). Overall (OS) and event free survival (EFS) at five years from DLT are shown in table 1. Outcome was significantly superior in BMT recipients, in particular if DLT was given for hematological, rather than cytogenetic or molecular relapse. In a cox regression model, four factors were associated with better OS/EFS: Transplantation in CP1 (p<.001/.002), early stage at relapse (molecular/cytogenetic vs. hematological, p<.00/<.001), a longer remission after transplantation (p=.005/.028), and BM vs. PBSC for transplantation (p= .003/.004). In contrast, age of patient and donor, acute or chronic GvHD after transplantation, the use of chemotherapy or imatinib before DLT, and the initial number of transfused CD3+ cells were not significant.

The results of this retrospective study suggest a reduced efficacy of DLT in CML relapse following related allogeneic PBSCT as compared to BMT. This effect seems to be independent from established risk factors as disease stage and remission duration. However, since several adverse factors are not equally distributed among the two groups, a bias might be induced, although no statistically significant imbalance could be detected. Therefore, the observation needs to be confirmed in a larger cohort or a prospective study.

Table 1. Outcome after DLT No BMT PBSCT p Total 346 260 86 OS at five years from DLT 71% 75% 66% < .001 EFS at five years from DLT 61% 64% 41% < . 001