bluuebunny/crossref_metadata_2025 huggingface dataset:
In Vivo Evaluation of a BAFF Inhibitor AMG 523 Suggests Lack of Efficacy in Multiple Myeloma Tumor Models.
Authors: Xu Jiangchun , Leenheer Evy D. , Piasecki Julia , De Vries Anne-Renee V. , Vanderkerken Karin , Croucher Peter
Published: April 2020

B-cell activating factor (BAFF), a member of the TNF superfamily, has been reported to play a role in the growth and survival of B-cell malignancies. However, the effect of inhibiting BAFF in vivo on tumor growth has not been fully determined. Additionally, studies so far have not addressed relative contributions of BAFF vs. APRIL towards cancer growth as the inhibitors used, such as TACI-Ig and BCMA-Ig, inhibit both BAFF and APRIL ligands. In the present study, we examined the potential therapeutic utility of a BAFF inhibitor, AMG 523, for treating B-cell malignancies in murine multiple myeloma in vivo models. AMG 523 is a peptibody that is a potent BAFF antagonist as a result of its high affinity binding to BAFF (KD of 1 pM). It inhibits BAFF-mediated increases in human and mouse B cell proliferation (IC50 of 0.1 nM at 0.2 nM BAFF). To determine the in vivo efficacy of AMG 523 in myeloma cells, we used the 5T series of murine models of myeloma. The 5T murine models of myeloma are myelomas that arose spontaneously in an inbred substrain of C57 black mice (C57BL/KaLwRij substrain). Several of the 5T myeloma lines closely mimic myeloma disease in humans. AMG 523 was first evaluated in a 5TGM1 syngeneic in vivo myeloma model. 5TGM1 was injected into the C57BL/KaLwRij mice and mice were treated with 5 or 20mg/kg of AMG 523 three times a week for four weeks. Tumor burden was determined by measuring the serum level of IgG2b, a paraprotein secreted by 5TGM1 myeloma cells. AMG 523 decreased serum IgG2b levels at early time points (day 8 and 15) but not at late time points (day 22 and 27) compared to the control. AMG 523 was also tested in two bone microenvironment dependent murine myeloma in vivo models: 5T33MMvv and 5T2MM. Both 5T33MMvv and 5T2MM cell lines express TACI receptor but not BCMA. BAFF increased survival of 5T2MM cells ex vivo, but inhibiting the activity of BAFF in vivo with AMG 523 had no effect on tumor burden, measured by proportion of bone marrow occupied by 5TMM cells, in both 5T33MMvv and 5T2MM models. Taken together, our data suggests that the BAFF inhibitor AMG 523 lacks efficacy in murine multiple myeloma in vivo tumor models.
A Novel Notch Pathway Inhibitor Blocks Osteoclast Activity and Synergistically Induces Apoptosis with the Proteasome Inhibitor Bortezomib in Multiple Myeloma Cells.
Authors: Jundt Franziska , Schwarzer Rolf , Kaiser Martin , Acikgoez Oezlem , Heider Ulrike , Mathas Stephan , Preissner Robert , Sezer Orhan , Dorken Bernd
Published: April 2020

Notch pathway inhibition in multiple myeloma (MM) cells is a promising new therapeutic approach since it controls myeloma cell growth as we previously demonstrated (Blood. 2004; 103:3511–3515). Notch signaling is involved in the tight interactions between myeloma cells and the bone marrow microenvironment and induces tumor cell growth in MM. We provided evidence that Notch receptors are expressed on MM cells and that Notch ligands on MM and bone marrow stromal cells activate Notch signaling through homotypic as well as heterotypic interactions in MM cells. In this study, we analyzed whether Notch signaling might be activated in osteoclasts, which express Notch receptors but not the ligands. To that end, we co-cultured MM cells and human osteoclasts, which were generated from mononuclear hematopoietic precursors of healthy donors using in vitro RANKL/M-CSF stimulation. Co-cultivation specifically activated Notch in osteoclasts and induced osteoclast activity as measured by mRNA expression of the tartrate-resistant acid phosphatase. The novel Notch pathway inhibitor, so called γ-secretase inhibitor 1 (GSI1), that we recently identified by structural comparison of known inhibitors with unknown compounds by data bank screening, specifically inhibited Notch signaling in osteoclasts and blocked their activity in this co-cultivation assay. In addition, GSI1 induced apoptosis in osteoclasts in higher concentrations. We suggest from our data that GSI treatment controls MM cell growth and concomitantly aberrant osteoclast activity in vitro and possibly in vivo, that is under current investigation. We further hypothesized that GSI1 can be combined with the proteasome inhibitor bortezomib, which has been known to have in vitro and in vivo activity against MM. We evaluated the activity of the combination of GSI1 and bortezomib against MM cell growth and survival. Proliferation of MM cell lines treated with GSI, bortezomib and their combination was determined by CellTiter-Glo® luminescent cell viability assay. AnnexinV-FITC/PI staining and cleaved poly (ADP-ribose) polymerase (PARP) staining were used to determine the degree of apoptosis. Although treatment of MM cell lines (OPM2, LP1) with either drug alone significantly inhibited proliferation and induced apoptosis with concentrations of GSI1 (30–60 μM) and bortezomib (1–4 nM), the combination resulted in synergistic inhibition of cell growth and survival. Our data suggest that combination of GSI1 and bortezomib is a rational novel treatment option in MM that simultaneously targets different proliferative and anti-apoptotic pathways. Whether this combination might also have synergistic activity against aberrant osteoclast activity in MM will be further investigated.
Inducible Expression of Leukemia-Associated Shp2 (Ptpn11) Affects Multiple Stages of Hematopoiesis and Causes a Fatal Myeloproliferative Disorder (MPD) in Mice
Authors: Chan Gordon , Kalaitzidis Demetrios , Mohi M. Golam , Yang Wentian , Kutok Jeffery , Neel Benjamin
Published: April 2020

Germ-line mutations in ptpn11, which encodes the protein tyrosine phosphatase Shp2, cause ∼50% of Noonan Syndrome (NS), which is associated with an increased risk of juvenile myelomonocytic leukemia (JMML). Somatic Shp2 mutations are found in ∼35% of sporadic JMML; nearly all other cases have either activating Ras mutations or homozygous Nf1 deficiency. Shp2 mutations are also found at lower incidence in acute myeloid leukemia (AML), myelodysplastic syndrome (MDS), and B-cell acute lymphoblastic leukemia (B-ALL). NS and leukemia-associated Shp2 mutations can affect the same residue, but result in different substitutions. We showed previously that the leukemia-associated mutants E76K or D61Y, but not wild type (WT), Shp2 transform bone marrow (BM) or fetal liver cells. Transplantation of E76K- or D61Y-transduced BM evoked invasive MPD in ∼60% of recipients, with most dying at 6–7 months. In this model, mutant Shp2 is expressed under retroviral promoter control and the phenotype is variable and incompletely penetrant. To assess the effect of expressing a leukemogenic Shp2 allele under endogenous promoter control, we generated knock-in mice that can conditionally express Shp2D61Y (LSL-Shp2D61Y). Global expression of the D61Y heterozygous allele was embryonic lethal. Post-natal expression of D61Y, induced by treating Mx-1 Cre; LSL-Shp2D61Y mice with pI-pC, evoked fatal MPD with ∼50% mice dying within 5–7 months. Mutant mice showed a marked increase in WBC, expansion of the Gr-1+/Mac-1+population in BM and spleen, and histopathological evidence of infiltrating MPD. Unlike in our retroviral transduction/transplant model (and similar to JMML patients), most induced LSL-Shp2D61Y mice were also anemic. Mutant mice exhibited a marked depletion of quiescent LSK (Lin−Sca1+cKit+) cells in the BM, with a concomitant increase in LSK cells in the spleen. Interestingly, Shp2 mutant-expressing BM failed to promote long term reconstitution in BM transplant assays. Cells from mutant spleens did have some long term multi-lineage reconstitution activity, but the level was substantially less than predicted by their LSK content. Moreover, no transplant recipients develop MPD. In contrast to these effects on the stem cell compartment, D61Y directly allowed cytokine-independent differentiation of CMP and GMP in vitro. Macrophages from mutant mice showed enhanced GM-CSF-mediated proliferation and ERK activation. Our results showed that expression of leukemia-associated Shp2 at endogenous levels is sufficient to evoke MPD and has cell type-specific effect on different stages of hematopoiesis.
Characterization of del20q in Peripheral Blood of MPD Patients Using Copy Number Analysis and High Resolution Oligonucleotide CGH Array
Authors: Schaub Franz , Tiedt Ralph , Hermouet Sylvie , Girodon François , Kralovics Robert , Tichelli André , Skoda Radek C.
Published: April 2020

Previous studies showed that deletions of the long arm of chromosome 20 (del20q) are the most common chromosomal aberration in myeloproliferative disorders (MPD) and can be found in up to 10% of patients with polycythemia vera (PV). We screened 447 MPD patients (177 PV, 228 essential thrombocythemia (ET), 42 primary myelofibrosis (PMF)) for loss of gene copy number in the del20q region. Granulocyte DNA was assayed using a quantitative PCR assay for two different exons of the L3MBTL gene, which is located in the common deleted region. Since deletions can only be detected if the majority of granulocytes have lost a copy of L3MBTL, this method selects for del20q events that provided a selective advantage for the clone. Samples that showed a decrease in copy number in the initial screen were validated using primers in neighbouring genes. We found del20q in peripheral blood of 29/447 patients (6%), including 9/177 PV (5%), 12/228 ET (5%), and 8/42 PMF (19%). The observed frequency of del20q in peripheral blood is lower than reported in previous studies for bone marrow analysis. The patients positive for del20q in this screen were chosen for mapping of the deleted region by high density oligonucleotide comparative genomic hybridisation (CGH) array. A custom chip was designed with 94’000 probes from chromosome 20q, with an average resolution of 500bp. We have so far analyzed 6 patients and confirmed del20q by CGH in all of them. The deleted regions in these patients overlapped with the published common deleted region. We will use CGH to map the remaining 23 patients. Since the majority of the del20q positive patients also carried the Jak2-V617F mutation, we performed clonogenic assays and analyzed single colonies. In one patient all 94 colonies were double positive for del20q and JAK2-V617F. In a second patient 54 colonies were double positive and 10 colonies only carried del20q, but not JAK2-V617F, indicating that del20q occurred before the acquisition of JAK2-V617F.
Association between Polymorphic Variation at the IL5RA Locus and the Severity of FIP1L1-PDGFRA Positive Chronic Eosinophilic Leukaemia
Authors: Burgstaller Sonja , Kreil Sebastian , Waghorn Katherine , Metzgeroth Georgia , Preudhomme Claude , Zoi Katerina , White Helen , Cilloni Daniela , Zoi Christine , Brito-Babapulle Finella , Walz Christoph , Reiter Andreas , Cross Nicholas C.P.
Published: April 2020

FIP1L1-PDGFRA is a cytogenetically cryptic fusion gene seen in a minority of patients with chronic eosinophilic leukaemia (CEL). Observations in mice have suggested that FIP1L1-PDGFRA alone is insufficient to induce all features of the human disease, and that a secondary event associated with IL-5 overexpression may be required for the full phenotype. In this study we have investigated the hypothesis that constitutional genetic variation in IL-5 signalling may be associated with the development or severity of FIP1L1-PDGFRA positive CEL in humans. We genotyped six single nucleotide polymorphisms (SNP) within or close to the IL5RA or IL5 genes in 82 European patients with FIP1L1-PDGFRA positive CEL plus, as controls, healthy individuals (n=100), patients with FIP1L1-PDGFRA negative eosinophilia (n=100) or patients with chronic myeloid leukaemia (n=100). We found no association between SNP allele frequency between FIP1L1-PDGFRA positive and control cases. However for FIP1L1-PDGFRA cases, we found an association between the genotype at rs4054760, a SNP in the 5′UTR of IL5RA, and peripheral blood eosinophil count (P=0.026) as well as the presence or absence of tissue infiltration (P=0.032). Although these associations fell below the level of significance once corrected for multiple testing, no such association was seen in FIP1L1-PDGFRA negative cases and no difference in allele frequencies for rs4054760 was seen in control populations across Europe. Furthermore, in an analysis of 128 patients with CML, there was a trend towards lower expression of IL5RA expression in cases with a c/t or t/t genotype compared to those who were c/c (P=0.08). These data suggest that the constitutional IL5RA genotype may be associated with the severity of FIP1L1-PDGFRA disease.
Comprehensive Highly Multiplexed Single-Tube Liquid Bead Microarray Assay for Constitutively Activated Tyrosine Kinases in Chronic Myeloproliferative Disorders.
Authors: Roberts Mark J. , Zhou Yening , Miller Kenneth B. , Pihan German A.
Published: April 2020

Chronic Myeloproliferative disorders (CMPDs) are malignancies characterized by excessive proliferation of one or more of the myeloid lineages driven by constitutively activated tyrosine kinases (CATKs). CATKs are increasingly serving as targets for cancer-specific therapy and newer and more potent TK inhibitors are becoming available for clinical use. CATKs in CMPDs may result from karyotypically evident or cryptic chromosomal translocations leading to TK gene fusions or from karyotypically silent gain-of-function point mutations. More than ten CATK genes have been shown to result in CMPDs by imparting a demonstrable positive growth drive. CATK inhibitors important in the treatment of CMPDs have different inhibitory profiles, making it critical to identify the specific mutation involved in patients receiving targeted therapy. Of these TK genes, ABL, PDGFRB, PDGFRA, FGFR1, ETV6, JAK2 and MPL are the most frequently involved. Since most of these TK genes can participate in chromosome translocations with a large number of partner genes, the resulting number of possible CATKs is rather large, making detection and identification a challenging task. To date, two classic (B2A2 and B3A2) and seven less common fusions of the ABL and BCR genes have been described. In addition, over twenty non-BCR-ABL mutations have been shown to have pathogenetic relevance in CMPDs. We have developed a simple, high-throughput assay with a fast turnaround time that solves the complex problem of detecting and identifying multiple translocations and point mutations. Two steps are involved in this expandable assay. The first is a multiplex RT-PCR reaction that amplifies fusion transcripts and point mutations described in CMPDs from peripheral blood or bone marrow. This PCR reaction employs internal specific primers targeted to each of eight BCR-ABL mutations (E1A2, E1A3, B2A2, B3A2, E6A2, E8A2, E15A2, E19A2) eight non-BCR-ABL mutations (FIP1L1-PDGFRA, BCR-PDGFRA, BCR-FGFR1, MYO18A-FGFR1, CDK5RAP2-PDGFRA, ETV6-PDGFRB, RABAPTIN-5-PDGFRB, H4-PDGFRB) and two gain of function mutations (JAK2V617F and MPLW515L) and a biotinylated common primer set to increase amplification efficiency. The second is a liquid bead microarray (Luminex) which uses optically encoded beads to detect probes specific for multiple known translocations in a single tube. To date, we have selectively detected four BCR-ABL and three non-BCR-ABL mutations from cloned patient samples and the cell lines SupB15 (E1A2 p190 BCR-ABL fusion), K562 (B3A2 p210 BCR-ABL fusion) and BV173 (B2A2 p210 BCR-ABL fusion). Dilution studies using these cell lines demonstrate detection down to between 1/1000 and 1/100000 cell equivalents without significant cross-talk.

Figure Figure Figure Figure
Transformation to Myelofibrosis and Blastic Transformation of PV Are Associated with Higher JAK2 Mutation Levels.
Authors: Koren-Michowitz Maya , Amariglio Ninette , Landman Joseph , Rechavi Gideon , Ribakovsky Elena , Cohen Yoram , Nagler Arnon
Published: April 2020

The JAK2 V617F mutation is found in 80–97% of polycythemia vera (PV) pts. The aim of the current study was to correlate the JAK2 mutation level with clinical symptoms and complications including vascular events and late hematological complications. Thirty-nine JAK2 V617F positive PV pts were included; M-19, F- 20; mean age at diagnosis was 55 (range 25–89) years. JAK2 mutation was analyzed in the PB using a quantitative MALDI-TOF based assay (Leuk Res 2007) after a mean of 130 (range 0–429) months from diagnosis. There was no correlation between the JAK2 mutation level and the time elapsed between the diagnosis and JAK2 mutation analysis. The mean JAK2 mutation level was 59% (range 2.5–95%). A negative correlation was found between the JAK2 mutation level and the pts age both at diagnosis (r=−0.3439, p=0.0321) as well as at the time of analysis (r=−0.3285, p=0.0412). JAK2 mutation levels were higher in pts with late hematological complications including transformation to myelofibrosis and blastic transformation compared to those without hematological complications (mean mutation level 77% vs. 51%, respectively p=0.0019). The best cut off value for the development of late hematological complications was above 50% (p=0.0336) which suggests the presence of homozygous JAK2 clones in pts that develop these complications. Similarly, there was a trend towards higher mean mutation levels in symptomatic pts (69% vs. 54%, p=0.096), pts with splenomegaly (62% vs. 47%, p=0.077) and pts that received cytoreductive therapy (63% vs. 47%, respectively p=0.077), while there was no difference between the levels of mutation in pts with thrombotic events compared to those without thrombotic complications. Our findings of:

association between the JAK2 mutation level and late hematological complications and no association between the JAK2 mutation level and thrombotic events concur with the results of Vannucchi et al (Blood 2007) that reported on a higher rate of transformation to myelofibrosis in PV patients with homozygous JAK2 status and no association between homozygosity for the JAK2 mutation in PV and thrombotic events.

In conclusion, we found that late hematological complications including transformation to myelofibrosis and blastic transformation in PV pts are associated with higher JAK2 mutation levels while thrombotic events are not associated with higher JAK2 levels. These results may be of clinical and therapeutic significance pending their further confirmation by a larger cohort of patients.
Both Discordant and Concordant Bone Marrow (BM) Involvement Predict for a Poorer Outcome Independent of the IPI in Patients with Diffuse Large B-Cell Lymphoma (DLBCL) Treated with R-CHOP.
Authors: Sehn Laurie H. , Chhanabhai Mukesh , Singh Suman , Saville Wayne , Matso Dan , Spinelli John J. , Connors Joseph M. , Gascoyne Randy D.
Published: April 2020

Recently, it has been suggested that concordant but not discordant BM involvement is a negative predictor of outcome in DLBCL patients treated with CHOP (Chung et al, Blood 2007). The significance of BM involvement in pts treated in the current era of therapy including rituximab has not been fully examined. We evaluated the prognostic impact of BM involvement in DLBCL patients treated with the current standard of care, R-CHOP.

Patients: We identified 282 patients with biopsy proven de novo DLBCL treated in British Columbia (BC) with an R-CHOP regimen between 01/01/2001 and 01/01/2005 with complete clinical information and staging bone marrows available for review. Cases were identified using the Lymphoid Cancer Database of the BC Cancer Agency. Median follow-up is 44 mos (range 1–77).

Results: 234/282 (83%) had a negative staging BM, 27 pts (10%) had a positive BM with concordant histology (Con-BM) and 21 (7%) had a positive BM with discordant histology (Dis-BM) with predominantly small B-cells present. Clinical characteristics for the entire cohort were as follows: median age 64 y (range 18–88); 66% male; 65% stage III/IV; 39% PS>1; 51% elevated LDH; 31% >1 extranodal site. IPI risk factors: 10% 0; 42% 1–2; 48% 3–5. In addition to higher stage and greater extranodal involvement, pts with a positive BM were more likely to have a higher LDH and a poorer PS than pts with a negative BM. Compared to pts with Con-BM, pts with Dis-BM were more likely to be elderly, but otherwise exhibited a similar distribution of clinical prognostic factors. The Kaplan-Meier 4-year PFS was significantly worse for pts with both Dis-BM (32%) and Con-BM (46%) involvement compared to pts with a negative BM (75%) (p<0.0001) (see Figure). Similarly, 4-year overall survival was significantly worse for pts with Dis-BM and Con-BM involvement (53% and 54%) compared to pts with a negative BM (74%) (p=0.005). All patients with Dis-BM involvement who developed progressive or relapsed disease were believed to have progression of their aggressive lymphoma based on clinical behavior or biopsy proof. In a multivariate analysis controlling for the IPI, BM involvement (Dis-BM and Con-BM) remained an independent predictor of PFS (p=0.03).

Conclusions: In DLBCL pts treated with R-CHOP, both discordant and concordant BM involvement predict for a poorer outcome independent of the IPI. It is possible that pts with DLBCL and discordant BM involvement represent a subset of pts with transformed disease which is inherently less treatment-sensitive.

Figure Figure
NK and T-Cell Status as Well as Their Perforin Expression Is Related to Clinical Severity and Outcome in Difuse Large Cell Lymphoma.
Authors: Gutierrez Antonio , Rodriguez Jose , Martinez Jordi , Ballester Carmen , Crespi Catalina , Bea Maria Dolores , Amat Juan Carlos , Villavicencio Roberto , Morey Miguel , Duran Maria Antonia , Linares Cristina , Rifa Julio , Bautista Antonia , Voegler Oliver , Alemany Regina , Sampol Antonia , Besalduch Joan
Published: April 2020

A variable degree of immune impairment is present in lymphomas, either as a cause or a consequence of the disease. There is preliminary evidence that this immune status is related to disease control and survival. Our objective was to analyze the relationship of the immune status with the main clinical prognostic variables in DLCL. Seventeen patients with DLCL de novo were prospectively included in the study, receiving an homogenous induction treatment based on CHOP-R regimen. Median age was 60 (29–74); ECOG PS > 1 in 65%; Ann-Arbor stage > II in 53%; B-symptoms in 47%; more than one extranodal site in 47%; bulky mass present in 59%; high LDH in 65%; high beta-2-microglobulin in 56%. Levels of B, T and NK subpopulations were assessed at diagnosis by flow cytometry, including general populations as well as cytotoxicity, costimulation and adhesion phenomena. This was compared with main clinical prognostic variables included in the adjusted-IPI (a-IPI) and MD. Anderson Tumor Score (TS) by U of Mann-Whitney non parametric test or Kaplan Meier survival analysis. Worst a-IPI patients (2–3 scores) (59%) showed significant lower levels of total lymphocytes as well as of the following populations:

General T lymphoid markers (CD2 and CD3); expression of adhesion molecules (CD3+CD11a+, CD4+CD11a+ and CD8+CD11a+); expression of costimulatory molecules (CD4+CD28+ and CD8+CD28+); cytotoxicity in CD56+ cells (CD56+ Perforin+).

Worst TS patients (3–4 scores) (59%) showed significant lower levels of the following populations:

Activated or regulatory lymphoid subsets (CD3+CD25+); expression of costimulatory molecules (CD4+CD152+ and CD8+CD152+); cytotoxicity in CD56+ cells (CD56+ Perforin+).

With a median follow-up of 16 months we observed a better overall survival and progression-free survival data in patients with levels above median of CD56+ and expression of perforin by T and NK cells. We conclude that worst prognostic groups of a-IPI and TS in DLCL are associated with a worst immune status, based on T and NK immune phenomena analysis. Perforin expression by NK and T CD56+ cells was associated with outcome in DLCL.
Role of C-MYC Rearrangement in Survival Status of HIV Positive and HIV Negative Patients with Diffuse Large B-Cells Lymphoma.
Authors: Zhang Ling , Luu Hung , Mirocha James , Lill Michael , Alsabeh Randa
Published: April 2020

Background: Diffuse large B-cell lymphomas (DLBCL) are the most common type of lymphomas and have heterogeneous clinical behavior. Three commonly dysregulated genes (BCL-2, BCL-6 and C-MYC) have been recognized in DLBCL based on molecular gene profile studies. The 8q24 C-MYC rearrangement is found in 10–15% of patients with DLBCL. C-MYC status in HIV+ and HIV− population with DLBCL is not well studied and the role of C-MYC in the survival status of these two group is worthy of exploration.

Design: Ninety-one patients with DLBCL diagnosed between 01/2005– 04/2007 were included in this study. Atypical Burkitt lymphoma according to WHO classification was excluded. Patients were divided into HIV+ (26 cases, average 44.5 ± 10.7 y/o) and HIV− (65 cases, average 65 ± 17.2 y/o) groups. Eight plasmablastic lymphomas (PBL) (7 HIV+ and 1 HIV−) were diagnosed. FISH (Vysis, Des Plaines, IL) break-apart for the 8q24 C-MYC rearrangement (91 cases), immunohistochemical stains for CD20, CD3, BCl-2, CD10, BCl-6, EBER in all cases, p53 (available for 78 cases), and proliferation rate by Ki-67 (available for 89 cases) were performed. Categorical variables were compared with the Fisher exact test. Numerical variables were compared by either the t test or the Wilcoxon rank sum test, as appropriate. The Kaplan-Meier method was used to estimate survival. The Log-Rank test was used to compare survival in the following groups: HIV+ vs. HIV−, C-MYC+ vs. C-MYC−, Ki-67 proliferation ≥ 90% vs. <90%, and various combined profiles.

Results: C-MYC rearrangement was detected in 13/26 (50%) HIV+ DLBCL and 9/65 (14%) HIV− DLBCL (P<0.001). Excluding the 8 PBL cases, C-MYC was found in 7/19 (37%) HIV+ DLBCL and 9/64 (14%) HIV− DLBCL (P=0.04). High Ki-67(≥ 90%) and p53 positivity were more often found in HIV+ DLBCL than in HIV− DLBCL (P=0.02 and P=0.001, respectively). A Kaplan-Meier Survival Curve demonstrated that patients with C-MYC+ DLBCL had worse survival than C-MYC− patient, regardless of their HIV status (P=0.031); HIV+ DLBCL patients have poorer survival than HIV− DLBCL ones (P=0.0007). The proliferation rate by Ki-67 (≥90% or <90%) did not affect survival (P=0.65). Survival was superior in the HIV−/C-MYC− group compared to the combined group of patients who were HIV+/C-MYC +, HIV−/C-MYC+ or HIV+/C-MYC− (P<0.0001, Figure 1).

Conclusion: high frequency of C-MYC rearrangement is identified in HIV+ patients with DLBCL in comparison to HIV− patients with the same disease. C-MYC rearrangement plays a critical role in survival regardless of HIV status. Although high Ki-67 proliferation was more often noted in HIV+ than in HIV− DLBCL, higher Ki-67(≥ 90%) did not correlate with a shorter survival in HIV+ DLBCL.

Survival by HIV and C-MYC Status Survival by HIV and C-MYC Status
Vaccination of Acute Myeloid Leukemia Patients with Dendritic Cells Electroporated with mRNA Encoding the Wilms’ Tumor Protein WT1: A Phase I/II Trial.
Authors: Berneman Zwi N. , Van Driessche Ann , Van de Velde Ann , Nijs Griet , Braeckman Tessa , Stein Barbara , Vermeulen Katrien , Pieters Katrien , Gadisseur Alain P. , Schroyens Wilfried A. , de Vries I. Jolanda , Price David A. , Van Tendeloo Viggo F.
Published: April 2020

To date, Wilms’ tumor protein (WT1) is acknowledged as a valuable target for active specific immunotherapy in several solid and hematological malignancies, such as leukemia. Preclinical data from our laboratory and that of Hans Stauss have already shown that WT1 RNA-electroporated dendritic cells (DC) stimulate WT1-specific T cells in vitro (Van Driessche A et al. Leukemia2005;19:1863–1871). Therefore, we started a phase I/II dose-escalation trial in which patients with acute myeloid leukemia (AML) in remission received intradermal injections with WT1 RNA-loaded DC. Feasibility, safety and immunogenicity of the vaccine were investigated. Seven patients received four biweekly DC vaccines. A delayed-type hypersensitivity (DTH) test was performed 2 weeks following the last vaccination. Patients underwent an apheresis and monocytes were isolated using CD14-labeled magnetic beads by CliniMACS. DC were generated in 6-day cultures in clinical-grade medium supplemented with serum, GM-CSF and IL-4 and maturated with PGE2 and TNF-a. Keyhole limpet hemocyanin (KLH) was added during maturation as a CD4+ helper antigen. Mature DC were harvested, electroporated with WT1 mRNA and used as vaccines. Patients were monitored for minimal residual disease (MRD) by analyzing WT1 RNA expression in peripheral blood by qRT-PCR. When the patient was HLA-A2+, tetramer staining was performed to detect WT1-specific CD8+ T cells. Before and after the vaccination cycle, peripheral blood was collected for immunomonitoring purposes. There was successful DC generation and vaccine production in all patients selected. No serious adverse events or toxicity was seen and all vaccinations were well tolerated. A decrease in WT1 RNA expression was observed during the course of the vaccination in 3/5 patients who had an increased WT1 mRNA level in peripheral blood at the start of DC vaccination. A vaccine-specific immune response was demonstrated in 7/7 patients by an in vivo DTH reaction both to KLH as well as to WT1. By tetramer analysis, detectable levels of WT1-specific CD8+ T cells could be demonstrated during the course of the vaccination both in the peripheral blood as well as in the expanded DTH-infiltrating T cells from the skin biopsies. Preliminary data from immunomonitoring in pre- and post-vaccination T cell samples from 3 patients show a mixed T helper (Th)1/Th2 response towards the KLH and the WT1 protein following vaccination. We conclude that vaccination of AML remission patients with WT1 RNA-loaded DC is feasible and safe. Furthermore, the vaccine elicits anti-vaccine T-cell responses in vivo and a decrease in WT1 RNA expression levels was observed during MRD monitoring in some vaccinated patients.
Cyclin D1 Polymorphisms as Predictive and Prognostic Molecular Markers in 90 Mantle Cell Lymphoma Patients Treated with R-HyperCVAD.
Authors: Thompson Michael A. , Kadia Tapan M. , Bekele B. Nebiyou , Wang Michael , Romaguera Jorge E.
Published: April 2020

Background Mantle cell lymphoma (MCL) is an aggressive lymphoma with poor prognosis. Prognostic and predictive molecular markers may aid in identification of patients that may benefit from clinical trials of novel therapies. Cyclin D1 is a highly specific marker of MCL. Alternative splicing in the cyclin D1 gene (CCND1) is modulated by a frequent A/G polymorphism located within the splice donor region of exon 4. CCND1 genotype is associated with the age of onset of HNPCC and with shortened event free survival and greater risk of local relapse in resectable non-small cell lung cancer.

Methods Samples were collected from 90 MCL patients treated with R-HyperCVAD from 1999 to 2002. The DNA was produced from peripheral blood lymphocytes and bone marrow using the Genomic extraction Kit (Promega, Madison, WI). The CCND1 exon 4 (codon 242) polymorphism was evaluated using pyrosequencing in the M. D. Anderson Cancer Center core facility as previously described (Kong et al. Cancer Res60:249–52, 2000). Genotype groups were correlated with clinical characteristics under an IRB approved protocol.

Results The CCND1 genotype frequency for 90 MCL patients was G/G 20%, G/A 54%, A/A 26%. Clinical characteristics were not statistically significantly different between genotype groups. 89 patients were evaluable for response. The ORR at 6 cycles was 100% for all subjects. The CR+CRu rates were G/G 95%, G/A 85%, A/A 96% (p=0.048). The PR rate was higher for patients with genotype G/A (15%) compared to G/G (6%) and A/A (4%). There were no statistically significant differences with respect to failure free survival (FFS) between groups (p=0.361), although there was a trend for a higher FFS in the A/A genotype group. In addition, overall survival (OS) was higher for A/A genotype patients at 61 vs. 49 months for the other genotypes (p=0.01).

Discussion In a previous study of 42 MCL patients the CCND1 genotype frequency was: G/G 31%, G/A 55%, and A/A 14% (Howe and Lynas. Haematologica86:563–9, 2001). They showed that CCND1 polymorphisms did not affect prognosis. However, the chemotherapy regimens were not defined in that study. In our study, a larger number of patients were treated homogeneously with the chemotherapy regimen of R-HyperCVAD. Response rates were high and differed slightly by CCND1 polymorphism. The OS was increased for the A/A genotype with a trend for higher FFS. The discrepancy with prior studies may be related to subject genotype frequencies, treatment regimen, and other factors. The CCND1 polymorphisms may have a different predictive yield with other treatment regimens - eg, bortezomib or mTOR inhibitors. Conclusions Response rates were high in all CCND1 genotype groups, but CR rates were lower in the G/A genotype patients. The OS was increased for the A/A genotype with a trend for higher FFS. Future studies should evaluate the role of CCND1 polymorphisms with other active agents against MCL.
SB939: A Potent and Orally Active HDAC Inhibitor for the Treatment of Hematological Malignancies.
Authors: Sangthongpitag Kanda , Stunkel Walter , Bonday Zahid Q. , Goh Kee C. , Wang Xukun , Wu Xiaofeng , Hu Changyong , Wang Haishan , Yu Niefang , Sun Eric , Entzeroth Michael , Kantharaj Ethirajulu , Chen Chien-Shing , Wood Jeanette M.
Published: April 2020

Histone deacetylases (HDACs) are emerging new molecular targets for cancer therapy. Small-molecule HDAC inhibitors have been developed and shown to induce tumor cell cytostasis, differentiation and apoptosis in experimental models and efficacy in clinical trials in various hematological malignancies following intravenous and/or oral administration. SB939 is a novel HDAC inhibitor with improved metabolic, pharmacokinetic and pharmacological properties compared to other HDAC inhibitors currently in clinical trials1. The objective of this study was to characterize the anti-tumor efficacy of SB939 in preclinical models of hematological malignancies. SB939 selectively inhibits HDAC class I and II isozymes, with Ki values ranging from 16 to 247 nM. It inhibited the proliferation of cell lines from various haematological malignancies, including leukemia, lymphoma and multiple myeloma with IC50 values ranging from 80 nM to 200 nM. It induced cell cycle arrest leading to apoptotic cell death in tumor cell lines as well as primary cells isolated from patients with acute myeloid leukemia (AML) or chronic myeloid leukemia (CML). SB939 has excellent pharmacokinetic properties and tolerability after oral administration in mice1. The oral anti-tumor efficacy of SB939 was evaluated in models of AML (MV4-11) and lymphoma (Ramos) with the tumors grown subcutaneously in nude mice. After daily oral treatment at 50mg/kg (21 days for MV4-11; 14 days for Ramos), SB939 significantly reduced tumor growth in both models (%TGI values were 116% and 100% respectively in MV4-11 and Ramos). In the MV4-11 model, SB939 induced complete tumor regression, in 6/10 mice. In conclusion, our data demonstrate that SB939 is a potent, orally active anti-tumor drug with potential for the treatment of various types of hematological malignancies. 1Kanda Sangthongpitag, Haishang Wang, Pauline Yeo, Liu Xin, Evelyn Goh, Lee Sun New, Peizi Zeng, Xiaofeng Wu, Changyong Hu, Tony Ng and Kantharaj Ethirajulu. ADME attributes of SB939, a best-in-class HDAC Inhibitor, and its PK/PD correlation in the Pharmacological Species. EORTC-NCI-AACR Symposium on Molecular Targets and Cancer Therapeutics Prague Congress Centre, 2006, Nov 7–10; Prague, Czech Republic, Abstract number 166
Activity and Mechanism of Action of AS1411 in Acute Myeloid Leukemia Cells.
Authors: Chen Weiwei , Sridharan Vijayalakshmi , Soundararajan Sridharan , Otake Yoko , Stuart Robert , Jones David , Fernandes Daniel
Published: April 2020

AS1411 is a 26 base unmodified DNA aptamer which is currently in a Phase II clinical trial for the treatment of acute myeloid leukemia (AML) and will shortly enter a Phase II trial in renal cell carcinoma (RCC). AS1411 has previously been shown to have cytostatic (a result of arrest in S phase of the cell cycle) and cytotoxic effects on tumor cells, but not normal cells. AS1411 binds to nucleolin, which is overexpressed in the cytoplasm and on the surface of tumor cells. Treatment of MV4-11 cells (an AML-derived cell line) with low micromolar concentrations of AS1411 results in cell growth inhibition and a reduction in viability; this effect becomes apparent after 48 hours incubation. Co-immunoprecipitation of nucleolin and AS1411 from MV4-11 cells treated with radiolabeled AS1411 confirmed previous work that AS1411 binds to nucleolin. In addition, confocal microscopy showed that nucleolin is present in the cytoplasm and the plasma membrane of MV4-11 cells. Incubation of these cells with AS1411 decreased nucleolin mRNA levels, and nucleolin protein was decreased in both cytoplasm and nucleus to approximately 10% of control levels after 96 hours. The reduction in nucleolin levels started to appear at around 48 hours and may reflect cells arresting in S phase of the cell cycle. Nucleolin binds to an ARE sequence in the 3′-untranslated region of Bcl-2 mRNA resulting in stabilization of the mRNA. Using gel shift assays with a radiolabeled fragment of Bcl-2 mRNA we show here that AS1411 interferes with this binding. In addition, levels of Bcl-2 mRNA in MV4-11 cells decrease to 10% of control levels after 48 hours incubation with AS1411 and the level of Bcl-2 protein also decreases in comparison to control cells. Finally, mononuclear cells isolated from blood samples of AML patients were incubated with AS1411 for 96 hours and viability determined. In all samples, AS1411 caused a reduction in cell viability at low micromolar concentrations, similar to those required to kill cell lines. This work therefore demonstrates that AS1411 has both cytostatic and cytotoxic effects on AML cells and underpins the dosing regime of AS1411 in the ongoing AML clinical trial.
Role of Microparticles in Thrombin Generation in Patients at Risk for Atherothrombosis.
Authors: Kaufmann Viktoria , Mager Jutta , Graf Markus , Geiter Sabine , Vetr Helga , Laemmle Bernhard , Redondo Maurice , Binder Bernd R.
Published: April 2020

We have shown recently (Hron et al. JAMA 2006) that measuring thrombin generation can identify patients at low risk for recurrent venous thromboembolism and that peak thrombin is a suitable parameter to differentiate low risk from high risk patients. In this study we used the same assay (Technothrombin®TGA) to analyze patients at high risk of atherothrombosis (a swiss study of patients with a history of myocardial infarction). Moreover, we investigated the influence of microparticles on thrombin generation in these patients. Thrombin generation was measured using Technothrombin®TGA. The reagent used contained low levels of phospholipid and 5 pM tissue factor. Platelet poor plasma from 178 patients (n=178, age =55±8 ys, male/female = 157/21) who had a previous myocardial infarction and 79 normal age and sex matched controls. (n=79, age = 55±8 ys, male/female = 70/9) was analyzed. Microparticle-free plasma was generated from PPP plasma by filtration through a membrane (0.2 μm pore size) using a standardized filtration device (Ceveron®MFU 500, Technoclone). In the control group the mean value for peak thrombin in platelet poor plasma was 252±7.7 nM (mean±SD). A significantly higher (p< 0,05) peak thrombin was found in the group of patients (355±49 nM) when patients without medication were analyzed. In patients on aspirin (n=109) the peak thrombin was not significantly different from untreated patients (297±10 nM) while in patients on oral anticoagulation (n=34) peak thrombin was significantly lower (225±27 nM). In the group of patients on oral anticoagulation also the lag period was significantly longer as compared to controls or patients without medication (21.9±2 minutes versus 14.8±0.2 minutes) whereas aspirin treatment did not affect the lag period significantly. After removing microparticles from the samples, the peak thrombin values between patient and control group were no longer significantly different. The difference between microparticle free plasma and platelet poor plasma was significantly higher (p<0.05) in the patient groups without therapy (304±39nM) and in patients on Aspirin therapy (257±23nM) as compared to the normal controls(182.6±10.4nM). These data indicate that thrombin generation is significantly increased in patients with previous myocardial infarction and that this increased thrombin generation is likely due to microparticles contained in these samples.
ADAMTS13 Antigen, Activity, and Autoantibody and ADAMTS13/fXI Complex Levels in Patients with Venous Thromboembolism.
Authors: Fryer Hugh J.L. , Grafos Nicholas , Guinto Enriqueta R. , Wu Jogin , Bing Bai , Greenfield Robert S.
Published: April 2020

ADAMTS13 controls thrombus formation in microvasculature by proteolysis of ultralarge von Willebrand Factor in plasma. Severe ADAMTS13 dysfunction or deficiency due to genetic or immunological causes can result in the life-threatening microangiopathic disorder Thrombotic Thrombocytopenic Purpura (TTP). Although it may also be involved in limiting thrombus growth in larger vessels, the role of ADAMTS13 in other thrombotic disorders has not yet been clearly established. In this study we determined the levels of ADAMTS13 antigen, activity, autoantibody, and ADAMTS13/fXI complex in plasma from patients with venous thromboembolism (VTE) and from normal donors. ADAMTS13 antigen was measured using IMUBIND® ADAMTS13 Antigen ELISA, ADAMTS13 activity using the ACTIFLUOR™ ADAMTS13 Activity (FRET-based assay), ADAMTS13 autoantibody titers using IMUBIND® ADAMTS13 Autoantibody ELISA, and complex using IMUBIND® ADAMTS13/fXI Complex ELISA. The various ADAMTS13 related markers were all found to be abnormal in patients with VTE (deep vein thrombosis and pulmonary embolism) as compared with normal donors. For normal donors, the mean ADAMTS13 activity is 740 ± 110 ng/ml, whereas in patients with VTE ADAMTS13 activity is significantly lower (524 ± 200 ng/ml, P<0.001). ADAMTS13 antigen levels, however, do not differ significantly for patients with VTE compared with normal donors. Receiver-operating characteristic analysis of activity/antigen ratios shows high sensitivity and specificity for patients with VTE. Although the biochemistry and in vivo biology of the ADAMTS13/fXI complex is not completely understood, the ADAMTS13/fXI complex is 3.8-fold higher in patients with VTE relative to normal donors (P<0.05). Using an upper autoantibody cutoff level of 14.8 AU/ml, a higher percentage of patients with VTE have elevated levels of autoantibodies (26/94 or ∼28%) compared with normal donors (1/47 or ∼2%). D-dimer plasma level is a strong negative predictor for VTE. A substantial number of patients without VTE exhibit D-dimer levels above the normal cutoff, which thus excludes this assay for the positive diagnosis of VTE. We correlated D-dimer levels with the various ADAMTS13 parameters. Elevated D-dimer in patients correlates with abnormal levels of activity, autoantibody, and complex. Donors with high D-dimer levels that do not exhibit symptoms of VTE do not have abnormal activity or complex levels, but do have elevated autoantibody levels. These data suggest that ADAMTS13 activity, autoantibody, and ADAMTS13/fXI complex are potential biomarkers for VTE and furthermore, suggest that ADAMTS13 may play a role in the initiation or progression of VTE.
Impact of the Factor II G20210A Variant and the Factor V G1691A on Recurrent Venous Thromboembolism in Children: An International Multicentre Cohort Study.
Authors: Young Guy , Friedrichs Fauke , Goldenberg Neil , Kenet Gili , Manco-Johnson Marilyn , During Christine , Nowak-Gottl Ulrike
Published: April 2020

The recurrence rate of thrombosis in children following a first thrombotic event ranges from 3% in neonates to 8% in older children. The relative importance of the factor II and factor FV mutations is unknown. We present a multicentre cohort study to assess the rate of symptomatic VTE recurrence per 1000 person-years in children heterozgous for the FII and FV mutations following a first VTE. Data were pooled to increase power for the secondary aims, e.g. time to recurrence, and predictors of recurrence. Between January 1994 and December 2006, 251 consecutively enrolled VTE patients aged newborn to ≤18 years (median 5.2 years: male n=141) carrying the FII (n=61) or FV mutation (n=190) were followed for a median of 58 (max 156) months. 128 of 251 VTE patients (51%) had at least one underlying medical condition at VTE onset, and 15 were heterozygous for both mutations. Children received acute anticoagulation (AC) with unfractionated heparin or low-molecular weight heparin, followed by AC with LMWH or warfarin for a three to 6 month period in 70% of cases. Of the 251 patients enrolled, 24 (9.5%: recurrence rate of 19.3 per 1000 person-years, 95% confidence interval (CI): 12.9–28.8) had recurrent VTE at a median (min-max) of 3.5 (0.1–120) months. Not including combined defects, the recurrence rate per 1000 person-years was 41.4 (95%CI: 22.3–77) for patients with the FII mutation, and 14 (95%CI: 8.3–23.6) for carriers of the FV mutation. Median (min-max) age at recurrence was 13.4 (0.1–17) years, 12 of 24 patients were male (50%), and in 21 of 24 children (87.5%) recurrence occurred after withdrawal of AC. When comparing FII with FV subjects, Cox regression analysis showed that the factor II mutation (HR/95%CI: 2.5/1.1–5.9; p=0.031) was associated more frequently with a second VTE. In addition, older age (> 2 years) at first VTE onset (HR/95%CI: 1.1/1.01–1.14; p=0.025) independently influences the second VTE event [3.5 years (non-recurrence) vs. 12.7 years (recurrence); p< 0.0001]. Among patients suffering from recurrent VTE, 58% occurred within the first six months following VTE onset. Time to recurrence (FII vs. FII and age at onset >/< 2 ears) is shown in the Kaplan Meier analyses. The overall VTE recurrence rate of 9.5% is within the range recently reported in children. However, when comparing FII and FV carriers, the factor II G20210A variant is more often associated with a recurrent VTE. In addition, independently from the underlying gene mutation age > 2 years at first VTE increase the risk of a second symptomatic venous thrombosis.

Figure Figure