bluuebunny/crossref_metadata_2025 huggingface dataset:
Delivery and Toxicity of Fludarabine-Based Combination Chemotherapy Regimens in Older Patients.
Authors: Polizzotto Mark N. , Tam Constantine S. , Januszewicz Henry , Prince Miles , Wolf Max , Seymour John F.
Published: April 2020

Fludarabine (F)-based combination chemotherapy regimens are highly effective in a range of indolent lymphoproliferative disorders. Despite the prevalence of such disorders in older patients, the deliverability of these regimes in patients aged >= 60 has not been assessed. We analysed the delivery and toxicity of three F-based regimens, all using F 25 mg/m2/dx3 q28d, in 82 adults aged >= 60 years, and compared this with the same regimens in 99 adults aged < 60. The sample comprised 66 patients (32 >= 60) treated with F and cyclophosphamide (C; 250 mg/m2/dx3); 29 with F and mitoxantrone (M; 10 mg/m2 x1; 12 >= 60); and 86 with FC and rituximab (R; 375 mg/m2 x1; 38 >= 60). 349 cycles in older patients were compared with 393 cycles in younger patients for haematologic nadirs, infectious complications and organ toxicity. Both groups received a median of 4 cycles, although older patients were more likely to require dose reduction (4.3% of cycles versus 1.2%, P < 0.001) and growth factor support (3.8% versus 1.8%, P=0.01). The cohorts were well matched for baseline characteristics other than age.

Overall, older patients had a slightly higher rate of infections (18%/cycle versus 15%/cycle), though this was not statistically significant (P = 0.28). For severe (grade >=3) infections the difference was minimal: 6% versus 7% (P< 0.5). The rates of neutropenia < 1.0 and 0.5 were 13% and 22% versus 11% and 20% for older and younger patients, respectively (all P values>0.1). The rates of thrombocytopenia < 100 and < 50 were 21% and 5% versus 16% and 5% for older and younger cohorts (all P values < 0.1). Other organ toxicities were uncommon, and showed no difference between age groups. Treatment-related mortality in both cohorts was <1% (P > 0.5).

Comparison within the cohort aged over 60 showed that those aged 70 and over were at higher risk of haematological and infectious toxicity. 82 cycles delivered to 23 patients aged >= 70 were compared with 267 cycles delivered to 61 patients aged 60 to 69. The rate of infection for those over 70 was 25% versus 16% in those aged 60 to 69, though this was not statistically significant (P=0.07). For severe infections (grade >=3), the rates were 13% versus 6% (P=0.03), while rates of neutropenia < 0.5 and thrombocytopenia < 50 were 32% and 15% versus 8% and 3% for those >= 70 and 60 to 69 respectively (all P values > 0.001).

These results demonstrate that F-based regimens are well tolerated and can safely be delivered to most older patients, with a modestly increased rate of infectious morbidity, but no increased treatment-related mortality. However, for patients aged >=70 the increased rate of toxicity mandates careful patient selection and monitoring.
Systematic Review of 18F-Fluorodeoxyglucose Positron Emission Tomography for Post-Treatment Evaluation of Malignant Lymphoma.
Authors: Zijlstra Josée M. , Hoekstra Otto S. , Lindauer-van der Werf Gerda , Hooft Lotty , Riphagen Ingrid I. , Huijgens Peter C.
Published: April 2020

Despite the increasing number of publications concerning 18F-Fluorodeoxyglucose Positron Emission Tomography (FDG-PET) for post-treatment evaluation of lymphoma and the increasing availability of this novel diagnostic modality, its exact role in response assessment after therapy is still unknown. The aim of this study was to systematically review the literature regarding the diagnostic performance of dedicated FDG-PET in evaluation of first line therapy of Hodgkin’s disease and (aggressive) non-Hodgkin’s lymphoma, and to calculate summary estimates of its sensitivity and specificity. The databases of PubMed and Embase were searched for relevant studies, up to January 2004. Criteria for inclusion of studies were 1) histologically proven Hodgkin’s disease (HD) or aggressive non-Hodgkin’s lymphoma (NHL), 2) evaluation of post-treatment patients following first line therapy, 3) the use of dedicated (ring) PET using 18FDG, and 4) study population of at least ten patients. Two reviewers independently assessed the methodological quality of each study. As a valid reference test, histology or follow-up of at least 12 months were accepted. A meta-analysis of the reported sensitivity, specificity, and positive Likelihood Ratio (LR+) and negative Likelihood Ratio (LR−) of each study were performed.

Fifteen studies, involving 705 patients, met the inclusion criteria. The studies had several design deficiencies. The majority of studies did not describe whether the reference test was interpreted without knowledge of the FDG-PET findings. Furthermore, in only two studies, patients entered the study consecutively. In all studies, there was a description of the spectrum of patients included, i.e. all patients for post-treatment evaluation or only patients with substantial residual masses post-treatment. Pooled sensitivity, specificity, LR+ and LR- for detection of residual disease in Hodgkin’s lymphoma were 84% (95% CI 71–92%), 90% (95% CI 84–94%), 5.5 (95% CI 3.4–8.7) and 0.25 (95% CI 0.1–0.55), respectively. For non-Hodgkin’s lymphoma, pooled sensitivity, specificity, LR+ and LR− were 72% (95% CI 61–82%), 100% (95% CI 97–100%), 36 (95% CI 11–125) and 0.30 (95% CI 0.21–0.42), respectively.

In conclusion, FDG-PET showed reasonable sensitivity and high specificity for evaluation of first line therapy in Hodgkin’s and in non-Hodgkin’s lymphoma.
Durable Remission with Decreased HTLV-I Proviral Load after Reduced-Intensity Stem Cell Transplantation (RIST) in Patients with Adult T-Cell Leukemia/Lymphoma (ATL).
Authors: Tanosaki Ryuji , Nosaka Kisato , Mineishi Shin , Mori Shin-ichiro , Kim Sung-Won , Kami Masahiro , Kannagi Mari , Sonoda Shunro , Okamura Jun , Fukuda Takahiro , Tobinai Kensei , Takaue Yoichi
Published: April 2020

Background. ATL is an HTLV-I-associated hematological malignancy. The majority of ATL patients are not curable using current chemotherapy with median survival time of 13 months. Recently, patients undergoing allo-SCT with conventional conditioning regimen were reported to have a 3-year overall survival (OS) rate of 45%, however, with high transplant-related mortality (TRM) rate as much as 40% (Fukushima T. Leukemia19:829, 2005). Since most ATL patients are over 50 years old with various complications, limited numbers of patients are eligible for conventional transplant. We have already conducted a phase I multi-center trial testing the feasibility of RIST in ATL patients, where 2-year OS was 33% and some differences in the outcome were suggested among participating institutes. Hence, we investigated the efficacy of RIST performed exclusively in our single institute (NCCH, Japan).

Patients and Methods. Between Oct 2000 and Jun 2005, 16 ATL patients underwent RIST from a related donor. Since 3 have been enrolled into an on-going multi-center trial, 13 patients were analyzed in this retrospective study, including 3 who were enrolled into the above-mentioned multi-center trial (Okamura J. Blood105;4143, 2005). Median age was 51 years old (range, 44–61), 8 males and 5 females, and 8 were of acute type and 5 of lymphoma. The disease status at transplant was 3 CR, 7 PR and 3 primary refractory. Three donors were HTLV-I healthy carriers. Eleven were HLA 6/6, 2 were 5/6, 12 were PBSCT and 1 was BMT. Conditioning regimen was fludarabine 30 mg/m2 x6, busulfan 4 mg/kg x2 with (n=5) or without (n=8) rabbit ATG 2.5 mg/kg x2. Prophylaxis of GVHD was CSP alone.

Results. Engraftment was rapid (median, 11 days) in all cases, and there was no TRM. Acute GVHD of grade II–IV developed in 7, and chronic extensive GVHD in 5. Eleven patients achieved CR after RIST, and 6 relapsed or progressed, among whom 2 achieved CR after cessation of CSP, donor lymphocyte infusion and local irradiation, and have been disease-free for more than 1 year. Ten patients are alive, 9 without disease, with median follow-up time of 26 mos (range, 4–45 mos). HTLV-I proviral titers determined using real time PCR decreased after RIST and became undetectable in 8 out of 13 patients. In 3, including one transplanted from an HTLV-I carrier donor, anti-HTLV-1 antibody transiently decreased and became nearly undetectable.

Conclusions. RIST is feasible and safe in ATL patients, and it has not only an anti-ATL but also an anti-HTLV-I activity. This is the first report documenting the efficacy of RIST for ATL in terms of survival and remission durability.

Figure Figure
Potent Cytotoxicity Against Multiple Myeloma and Acute Myeloid Leukemia Cells by a New Histone Deacetylase Inhibitor ITF2357.
Authors: Rambaldi Alessandro , Micò Caterina , Cuppini Lucia , Domenghini Marcello , Borleri Gian Maria , Barbui Anna Maria , Leoni Flavio , Mascagni Paolo , Salvi Anna , Golay Josee , Introna Martino
Published: April 2020

ITF2357 (Italfarmaco, Italia) is a novel hydroxamic acid-based HDAC inhibitor (HDACi) that has shown reduced toxicity in phase I studies. We have investigated the cytotoxic and anti-proliferative activities of ITF2357 in multiple myeloma (MM) and acute myelogenous leukaemia cells (AML). ITF2357 had a strong cytotoxic activity in 8/9 MM and 6/6 AML cell lines, with a mean IC50 of about 0.2 μM, a concentration largely attained following oral administration of safe doses of ITF2357 to healthy individuals. In contrast SAHA, the prototypic hydroxamic HDACi, showed an IC50 of about 1 μM or above in all cases. The cytotoxic activity of ITF2357 was due to induction of apoptosis, as documented by detection of annexin V and cleaved caspase 3. The ITF2357 induced hyperacetylation of histones in cell lines resistant or sensitive to the cytoytoxic activity of the drug is under investigation in order to further define its mechanism of action. ITF2357 had also more potent cytotoxic activity compared to SAHA against freshly isolated CD138+ purified MM cells and AML samples, with an IC50 of about 0.1 μM in 3/3 MM and 13/15 AML cases. Sensitive AML cases included five cases of FAB M1, five M2 and three M4. Three of the sensitive cases carried a t(8;21) translocation, 2 an inv(16), 6 had a normal and 2 a complex karyotype. Furthermore four of the sensitive AML also carried a flt3 internal tandem duplication and 3 a type A mutation in the nucleophosmin 1 gene (NPM1). The 2 more resistant AML cases (one M1 and one M5, both with normal karyotype) showed nonetheless a response to the drug with an IC50 of about 0.5 μM. We have also developed a culture system to grow freshly isolated AML cells for at least 3 weeks in vitro on human mesenchymal cells (MSC). Interestingly, ITF2357 was cytotoxic for primary AML cells stimulated to grow in optimal conditions on MSCs, at the same dose as in standard short term cultures. In contrast ITF2357 was not cytotoxic for MSCs even at the 1 μM concentration. The strong cytotoxic activity of ITF2357 on MM and AML has provided the framework for ongoing phase I studies of ITF2357 in these malignancies.
Raf Inhibitor BAY 43-9006 Induces Bim Dephosphorylation and Activates the Intracellular Apoptotic Pathway in AML.
Authors: Zhang Weiguo , Konopleva Marina , McQueen Teresa , Cortes Jorje , McCubrey James , Andreeff Michael
Published: April 2020

We have previously demonstrated constitutive activation of MAPK signaling in 70% of primary AML samples (Millela et al, JCI108:851–859, 2001), suggesting that upstream kinases (Raf and MEK) may play a role in the leukemic transformation of myeloid cells. BAY 43-9006 is a small molecule Raf kinase inhibitor that has demonstrated potent anti-tumor activity against solid human tumors in xenograft models. In this study, we tested the hypothesis that BAY 43-9006 inhibits leukemia cell growth and/or induces apoptosis by suppressing the activity of the MAPK pathway. In the in vitro kinase assay, BAY 43-9006 inhibited both Raf-1 and B-Raf-mediated MEK1 phosphorylation in a dose-dependent manner, with Raf-1 kinase being more sensitive to the inhibitory effects of BAY 43-9006 (IC50Raf-1, 1.37 μM vs. IC50B-Raf, 4.64 μM). BAY 43-9006 suppressed MEK1/2 and ERK phosphorylation in the AML cell lines OCI-AML3, HL-60, U937 and KG-1 in a dose-dependent manner after 24 hr treatment. Unexpectedly, BAY 43-9006 also inhibited AKT phosphorylation on Ser473 (after 4.5 hrs). BAY 43-9006 inhibited growth of AML cells in a dose- and time-dependent manner. The 50% inhibitory concentration (IC50) of BAY 43-9006 was 0.39, 1.14, 2.86 and 2.80 μM, respectively in OCI-AML3, HL-60, U937 and KG-1 cells after 72 hrs. This growth-inhibitory effect was mediated by a dose-dependent induction of cell cycle arrest in G1 mediated by the down-regulation of the cell cycle-related proteins cyclin E, cdk2 and cdc2, followed by induction of apoptosis after 72 hrs. In primary AML patient samples, BAY 43-9006 not only inhibited cell growth and induced apoptosis after 48–72 hrs in vitro, but also preferentially inhibited colony formation of AML progenitor cells compared to normal bone marrow cells [IC50: 2.33 μM vs. 9.34μM (CFU-GM), 5.69 μM (Erythroid) and 3.75 μM (Mixed), respectively]. Time-course analyses demonstrated that BAY 43-9006 suppressed phosphorylation of the pro-apoptotic protein Bim (at 4.5 hrs), caused loss of the mitochondrial membrane potential and cytochrome c release (at 6 hrs) followed by cleavage of caspases-3 and -9 but not of caspase-8, suggesting primary involvement of the intrinsic mitochondrial pathway. Furthermore, the pro-apoptotic proteins Bim and Bax were up-regulated after 48 hrs of BAY 43-9006 treatment, and the level of the inhibitor-of-apoptosis protein Survivin was down-regulated after 48 hrs. In summary, our data demonstrates that BAY 43-9006 inhibits Raf-MEK-ERK signaling and induces apoptosis in AML via Bim de-phosphorylation and activation of the intrinsic apoptotic pathway. The potential of BAY 43-9006 in the therapy of AML patients will be tested in a Phase I clinical trial.
Acquired Bortezomib (Velcade®)-Resistance Is Accompanied by Downmodulation of Proteasome Activity and Upregulation of Tripeptidyl Peptidase II (TPPII) and a Ubiquitin-Specific Protease.
Authors: Driessen Christoph , Gogel Jeanette , Kraus Marianne , Ovaa Huib , Beck Alexander , Rückrich Thomas
Published: April 2020

Because cell lines can be adapted to proteasome inhibition in vitro (Pfeifer G. et al., Science 283: 978–981, 1999.), secondary resistance of malignant cells towards Bortezomib (Velcade®) in vivo is a likely scenario. We have succeeded to adapt the human AML cell line HL-60 to Bortezomib in vitro, so that the adapted subline (HL-60a) shows normal viability and growth rate at 40 nM Velcade®, while cell death is induced above 10nM in the parental line. We hypothesized that alternative proteolytic pathways might allow the continued proliferation and survival of Bortezomib-adapted cells, and assessed the activity-profiles of proteasomal subunits, ubiquitin-specific proteases and lysosomal cysteine proteases in a functional proteomics approach, using recently developed synthetic affinity probes. These tools for the first time allow semiquantitative visualization of the individual members of these protease families, based on their activity, in contrast to western blot which lacks activity information or to the turnover of fluorogenic substrates, which is not truly protease-specific. After 72h of culture in Bortezomib-free medium (wash out phase), HL-60a cells contained significantly reduced levels of active proteasomal β1,β2, and β5 subunits, compared to the parental line, as confirmed by a reduced turnover of the β5-selective fluorogenic substrate Suc-LLVY-AMC. A panel of 7 different ubiquitin-specific proteases (USP) was visualized in both types of cells, using the affinity probe HAUbVS. Of these, a 97 kD USP that we have identified as USP14 by mass spectrometry-based sequencing, was significantly upregulated in HL-60a cells. By contrast, the activity of lysosomal cysteine proteases remained unchanged in HL-60a cells. Because the cytosolic protease tripeptidyl peptidase II (TPPII) can partially substitute for proteasome activity in lactacystine-treated cell lines, we assessed TPPII-activity using a fluorogenic substrate. We observed a significant upregulation of TPPII-activity in HL-60a cells, compared to non-adapted controls. Inhibition of TPPII, however, was not sufficient to restore Bortezomib-sensitivity in HL-60a cells. Interestingly, the combination of Bortezomib with the HIV protease inhibitor Ritonavir induced synergistic cytotoxicity both in HL-60 and HL-60a cells. Thus, Bortezomib-resistance is accompabied by upregulation of protease activities in alternative pathways. Combining different protease inhibitors like Bortezomib and Ritonavir might be a promising option to overcome primary or secondary Bortezomib resistance.
The Ellipticine Derivative NSC 338258 Has Anti-Myeloma Activity.
Authors: Tian Erming , Stephens Owen , Holbeck Susan , Barlogie Bart , Shaughnessy John
Published: April 2020

We recently discovered that amplification and overexpression of the CKS1B gene, which regulates the ubiquitination and proteasomal degradation of the cyclin-dependent kinase inhibitor p27Kip1, is linked to a poor prognosis in myeloma. We used the COMPARE algorithm (NCI, Developmental Therapeutics Program) to identify correlations between the expression of CKS1B in the NCI 60 cell line panel and the GI50 of nearly 50,000 anticancer compounds. This analysis revealed a strong correlation between CKS1B expression and the anticancer activity of Ellipticine and multiple derivatives of it. Ellipticine (5,11-Dimethyl-6H-Pyrido[4,3]Carbazole, MW=246.3), an alkaloid isolated from Apocyanaceae, is a topoisomerase II poison that induces topoisomerase II-dependent DNA cleavages. Previous studies have shown that the anti-neoplastic mechanism of Ellipticine is to form covalent DNA adducts mediated by human cytochromes P450. We obtained Ellipticine and twelve other CKS1B-correlated antineoplastic compounds from the NCI-DTP. In vitro analyses were carried out with 12 myeloma cell lines. Cell viability was detected using CellTiter-Glo Luminescent Assay (Promega, Co.). We found that one of the Ellipticine derivatives, NSC 338258 (EPED3) showed significant cell kill activity not observed with Ellipticine or the other compounds in all cell lines tested. In a dose response analysis of cell kill using 0.2, 0.02, and 0.002 uM EPED3, 12 out of 12 myeloma cell lines showed at least 50% reduction in cell viability within 24 hrs at 0.2 uM compared to no treatment controls. By 48 hours essentially all cell growth was inhibited. Only weak to moderate cell kill was noted at 0.02 and 0.002 uM at all time points tested. We next tested the activity of the topoisomerase II inhibitor VP-16-213 at the same concentrations and time periods and found only modest reduction of cell viability at day 5 at 0.2uM. Essentially no effect on cell viability was observed at 0.02 and 0.002 uM at any of the time points tested. We next tested whether EPED3 had a comparable anti-myeloma activity as Adriamycin at the same concentrations. Twelve myeloma cell lines were exposed to EPED3, VP-16-213, and Adriamycin at 0.2mM for a total of 5 days. Results of the mean relative luminescence showed that EPED3 promoted an immediate cell proliferation arrest within the first 24 hours of culture, with almost complete cell kill within 3 days. The other drugs were less effective with Adriamycin inducing a > 90% reduction only after 4 days and VP-16 a > 70% reduction at day 5. These data suggests that EPED3 may represent a potential new therapeutic for myeloma. Studies are currently underway to test the efficacy of EPED3 in our in-vivo SCID-Hu mouse model of primary myeloma and results of these studies will be presented.
Baicalein Inhibits IL-6-Mediated Multiple Signaling Cascades through Jak Family Kinase in Human Myeloma Cells.
Authors: Ma Zi , Liu Shangqin , Otsuyama Ken-ichiro , Kawano Michio M.
Published: April 2020

Chemoresistance is a major problem in the treatment of patients with multiple myeloma(MM). Interleukin 6(IL-6), the most important growth factor for myeloma cells, mediates multiple signals which play a central role not only in the cell survival and the proliferation but also in the chemoresistance. The agents that suppress the IL-6-mediated signaling have the potential for the treatment of MM, such as PS-341 and curcumin. We have previously shown that baicalein from Scutellaria radix inhibited the proliferation and induced apoptosis of human myeloma cells, partly due to the suppression of IkB phosphorylation, resulting in the downregulation of NF-kB activity (Blood105:3312,2005). In order to clarify whether baicalein could inhibit IL-6-triggered signaling cascades in myeloma cells, we first demonstrated that baicalein inhibited not only the activation of MAPK, but also the phosphorylation of Stat3 and Stat1 induced by IL-6. Baicalein could also inhibit the phosphorylation of Akt triggered by IL-6, but not IGF-1. Since gp130 is phosphorylated by Jak family kinase after IL-6 stimulation, we examined whether Jak kinases could be involved in the baicalein-induced blockade of IL-6-induced signaling mediating the survival and growth of myeloma cells and the drug-resistance. Furthermore, we showed by phosphoprotein immunoblotting analysis that baicalein inhibited the activity of Jak2 and Tyk2 in myeloma cell lines (U266 and NOP-2 cells). Therefore, our results indicate that baicalein can also inhibit IL-6-induced signaling through Jak family kinase in myeloma cells, and combined with its suppressive effect on NF-kB activity baicalein can lead to more effective suppression of the proliferation and survival of myeloma cells.
Melphalan Resistance in Multiple Myeloma Can Be Overcome by Proteasome Inhibition or Histone Deacetylation: Lack of Syngergism of the Combination.
Authors: Sonneveld Pieter , Kamst Eric , de Knegt Yvonne , Klarenbeek Naomi , Schoester Martijn
Published: April 2020

Multiple Myeloma (MM) is a disease of monoclonal plasma cells in the bone marrow which has a transient response to classic chemotherapy. At diagnosis, induction chemotherapy followed by high-dose melphalan (HDM) with stem cell support is used in most patients to achieve a clinical response. Because all patients will ultimately relapse, the treatment of melphalan-refractory disease represents a major clinical challenge and new agents are needed to overcome melphalan resistance. We have investigated the anti-myeloma efficacy of two new classes of targeted agents, i.e. proteasome inhibition and histone deacetylation inhibition alone or in combination in the melphalan sensitive MM1S and the Melphalan refractory MM1MEL2000 cell lines. The IC50 values of Bortezomib (B), Melphalan (M) and LAQ824 (L) in MM1S were 2.1 nM, 1.9 uM and 1.7 nM, respectively and in MM1MEL2000 3.9 nM, 50 uM and 4.0 nM. Using isobologram analysis a synergysm between B and L was observed in the sensitive, however not in the melphalan refractory cell line. These data indicate that B proteasome inhibition and histone deacetylation inhibition may be effective ways to overcome melphalan resistance. However, the previously reported synergism between these drugs does not seem to occur in melphalan resistant cells. The gene expression profiles of these cell lines were analysed using the Affymetrix U133plus 2.0 gene chip before and after treatment with melfaphalan or the proteasome inhibitor B or the histone deacetylation inhibitor L or the combination of B and L.

Genes that were highly expressed in the melphalan refractory derivate cell line MM1MEL2000 as compared with wild-type MM1S included GP M6B, ADAM23 and HTPAP. Following melphalan exposure, TMF1, a CEBp glucocorticoid interaction factor, WHSC1L1, a MMSET homologue with EGF like domain and several transcription factors had highly increased expression as compared to MM1S. With exposure to B combined with L, increased expression in MM1MEL2000 over MM1S was observed for GTP exchange factor TIAM1 which interacts with RAS and JNK, and the lymphoid enhancer factor, a notch transcription factor. It is concluded that Bortezomib and the histone deacetylase inhibitor LAQ824 are effective agents to overcome melphalan resistance in multiple myeloma. However, the combination fails to show the synergism observed in melphalan sensitive cells. Gene analysis sofar does not provide a clear explanation for this lack of synergism. A comprehensive summary of the observed shifts of gene expression profiles in melphalan resistant cells following exposure to these agents, will be presented.
Overexpression and Preferential Nuclear Translocation of the Transcription Factor Yin Yang 1 (YY1) in Human Bone Marrow-Derived Multiple Myeloma.
Authors: Huerta-Yepez Sara , Baritaki Stravoula , Hernandez-Cuecto Angeles , Anguino-Hernandez Yu-Mei , Neshat Mehran , Chen Haiming , Campbell Richard A. , Gordon Melinda S. , Berenson James R. , Bonavida Benjamin
Published: April 2020

Yin Yang 1 (YY1) is a ubiquitously expressed zinc-finger transcriptional regulator that can function as an activator, a repressor, or an initiator binding protein. YY1 has been implicated in the control of cell growth, development, differentiation and tumor suppression. We have recently reported that YY1 regulates the transcription of death receptors in carcinoma and lymphoma and thus, YY1 regulates tumor sensitivity to immune-mediated apoptosis (Huerta-Yepez, et al., 2004, Oncogene23: 4993–5003; Vega, M., et al., 2005, Journal of Immunology175: 2174–2183). In addition, we have also reported that human prostate tumor tissues overexpress YY1 (Seligson, et al., 2005, International Journal of Oncology 27:131). This study investigated the expression of YY1 in multiple myeloma (MM) with the objective of determining whether YY1 plays a role in the progression and drug- resistance of MM. We have initiated these studies by examining nine bone marrow (BM) samples derived from patients with MM. Immunohistochemical studies were performed for the detection and cytoplasmic or nuclear localization of YY1 in the MM cells and also in adjacent normal mature and immature cells. The intensity of staining by the anti-YY1 antibody was scored and the relative intensity was calculated. Two slides from each patient were analyzed and 200 cells per slide were scored. Mean intensities of all samples were calculated and the data were subjected to statistical analysis. YY1 expression in normal BM was low and primarily of cytoplasmic origin. In contrast, YY1 was significantly overexpressed in MM cells. The mean intensity in the MM was approximately three-four fold higher than that of the normal cells and was primarily of nuclear origin (p-value < 0.05). The signals that control shuttling YY1 are undefined. The expression of YY1 in normal mature and immature cells was low and there was comparable staining in the cytoplasm and the nucleus. Analysis of the cell distribution expressing YY1 by flow cytometry revealed that greater than 50% of the cells in the CD38+ subset expressed YY1. In addition, the MM tumor cells also expressed high level of pleiotrophin (PTN) and the patients had high levels of circulating PTN. PTN is a growth factor for MM and PTN transcription is regulated by an initiator element and could thus be a target of YY1-mediated transcriptional control. These findings suggest that YY1 overexpression may be involved in the pathogenesis of MM and is a potential target for therapeutic intervention. Current studies are aimed at examining the prognostic and diagnostic roles of YY1 and PTN in MM and molecular mechanisms that underlie their overexpression in MM.
Measurement of HSP90 and HSP70 in CD34-Positive Cells: Lower Levels in Myelodysplastic Syndrome Than in Acute Myeloid Leukemia and Correlation with Clinical Behavior.
Authors: Gorre M. , Jilani I. , Chang R. , Chan H. , Urcia R. , Keating M. , Kantarjian H. , Estey E. , O’Brien S. , Giles F. , Albitar M.
Published: April 2020

Heat shock proteins (HSPs) are molecular chaperones involved in binding and regulating the levels of several client cellular proteins. HSPs are induced by stress and play a role in the modulation of apoptosis and proliferation. Expression of HSPs by acute myeloid leukemia (AML) cells has been reported to correlate with more aggressive disease. Here we used a flow cytometry approach to quantify the expression of HSP90 and HSP70 in specific cell populations in bone marrow. Quantification using QuantiBRITE and PE (phycoerythrin)-labeled antibodies with a 1:1 ratio allowed us to specifically measure the antibody binding capacity in 100 CD34+ cells (molecules/100 CD34+ or CD3+ cells). Using this approach, we compared bone marrow samples from patients with myelodysplastic syndrome (MDS) and patients with AML. This approach can also be used to monitor patients treated with therapeutic agents that target HSPs, such as 17-allylamino-17-demethoxygeldamycin (17AAG). The percentage of CD34+ cells expressing HSP90 (P=0.008) and HSP70 (P<0.001) was significantly lower in MDS (n=20) than in AML (n=33) patients. Similarly, the percentage of CD3+ cells expressing HSP90 and HSP70 was significantly higher in MDS than in AML patients (P<0.01). This suggests that the environment in the bone marrow (cytokines, chemokines, other factors) may affect the levels of HSPs in neoplastic and normal cells in a similar fashion. In patients with MDS, higher levels of HSP90 were associated with shorter survival (P=0.03). However, after achieving remission (CR), MDS patients who expressed high levels of HSP90 had significantly longer remission duration (CRD) (P=0.03). These findings not only confirm that the environment and blasts in patients with MDS are different from those in patients with AML, but also suggest that therapy targeting HSPs may be a rational approach in patients with MDS.

Figure Figure
Lenalidomide Selectively Inhibits In Vitro Growth of the Malignant Clone in Myelodysplastic Syndrome (MDS) Patients with 5q Deletion.
Authors: Jädersten Martin , Pellagatti Andrea , Forsblom Ann-Mari , Emanuelsson Emma K. , Merup Mats , Samuelsson Jan , Wainscoat James S. , Boultwood Jacqueline , Hellström-Lindberg Eva
Published: April 2020

The immunomodulatory drug lenalidomide induces 82% complete cytogenetic remissions in patients with myelodysplastic syndrome (MDS) and 5q31 deletion. Lenalidomide has multiple effects, but the causal mechanisms of action are unknown. In this study we assessed the direct effect of lenalidomide on hematopoietic cells from eight MDS patients with del(5)(q31) and from five healthy controls. Lenalidomide titrated up to 500 μM caused no growth inhibition of mononuclear cells from healthy controls. Selected CD34+ hematopoietic stem cells were then cultured with or without 10 μM of lenalidomide in a 14-day model for pure erythroblast differentiation in a medium containing IL-3, IL-6, and SCF, and with addition of Epo during the second week. Cell count and viability was monitored regularly, and FISH and FACS analyses were performed at day 0, 7, and 14. The median proportion of 5q- cells by FISH at day 7 was 98% (range 86–99), dropping to 88% (range 35–98) at day 14 due to a variable outgrowth of cytogenetically normal cells. Day 7 cells, the majority still being 5q-, were used for gene expression analyses. In erythroblast cultures with cells from healthy controls, lenalidomide had no inhibitory effect on fold increase of cell counts (P=0.92). However, in cultures with cells from 5q- patients, the clone with 5q deletion showed significant inhibition of fold increase at day 14 (P=0.009), while the cytogenetically normal progenitors were not inhibited (P=0.83). Gene expression profiling was performed using Affymetrix Human Genome U133 Plus 2.0 Arrays. A group of genes was found whose expression was affected by the addition of lenalidomide to the cultures of both normal erythroblasts and 5q- erythroblasts. Furthermore, lenalidomide decreased the proportion of cells expressing late erythroid markers on FACS analysis at day 14. We conclude that lenalidomide selectively inhibits in vitro growth of 5q- hematopoietic stem cells, while not affecting growth of cytogenetically normal cells from MDS patients with 5q deletion or from healthy controls. In addition, we see that lenalidomide affects cell differentiation and induces changes in gene expression.
Identification of Pathogenetically Important Genes within the Common Deleted Region (CDR) of the 5q- Syndrome.
Authors: Lehmann Soren , Raynaud Sophie , Desmond Julian C. , Koeffler Phillip H.
Published: April 2020

The 5q- syndrome is characterized by refractory anemia, normal or high platelet count, hypolobulated megakaryocytes, a good prognosis and a low risk of leukemic transformation. Although the CDR has been defined to a 1.5 Mb interval on the long arm on chromosome 5 (5q33.1), the molecular pathogenesis of the disease is still unknown. The CDR contains 39 known-genes of which 33 have been shown to be expressed in hematopoietic stem cells. In order to elucidate the molecular mechanisms behind the 5q- syndrome, we performed real-time quantitative PCR on these 33 genes. Samples from the bone marrow of 12 patients with a sole deletion of 5q and 14 patients with MDS with normal karyotype were initially analyzed. The genes that showed the most pronounced decrease in expression in the 5q- samples were: SLC36A1 (89% down-regulated compared to non 5q-), G3BP (79%), ATOX1 (76%), CSF1R (76%), RPS14 (74%), PDGFRB (73%), TNIP1 (72%), SPARC (71%), ANAX6 (69%), NSDT (66%) and TIGD (60%). SPARC expression was found to be higher in both types of MDS samples compared to normal bone marrow (n=18) as well as compared to seven leukemic cell lines (HL-60, NB4, HEL, KG1, K562, U937 and TP-1). ATOX1 expression was highly over-expressed (20- to 80-fold) in the leukemic cell lines and modestly but significantly higher in normal bone marrow compared to both types of MDS. For G3BP, the expression was similar in normal bone marrow compared to the non-5q- samples but 1- to 10-fold higher in the cell lines. RPS14 was down-regulated in both types of MDS compared to normal bone marrow and leukemic cell lines. Thus, we have identified the most significantly down-regulated genes within the CDR of the 5q- syndrome. Based on our expression data, their known biological functions and on publicly available tissue expression data, genes such as G3BP, ATOX1, TNIP1, RPS14 and CSF1R are interesting targets for further studies. Biological studies are currently being performed on these genes with respect to their role during hematopoiesis with special focus on erythropoiesis.
TNF-α Stimulates Matrix Metalloproteinase Expression in Myelodysplastic Syndromes (MDS): Therapeutic Potential for Inhibitors of TNF-α and MMPs.
Authors: Janowska-Wieczorek A. , Marquez-Curtis Leah A. , Gan Kenton , Larratt Loree , Woods Anthony
Published: April 2020

MDS is characterized by ineffective hematopoiesis with increased intramedullary apoptosis which has been correlated in turn with high levels of tumor necrosis factor (TNF)- α. Previously, we showed that TNF-α strongly upregulates the secretion of the matrix metalloproteinases (MMP)-2 and -9 in normal CD34+ cells. Elevated expression of MMPs has been suggested to contribute to cancer progression and leukemic dissemination and MMPs also facilitate the secretion of TNF-α. In this work, we hypothesized that TNF-α-induced MMP production plays a role in the progression of MDS. We therefore examined the effects of recombinant human (rh) TNF-α on the expression and secretion of MMP-2, MMP-9 and membrane type (MT) 1-MMP (known activator of MMP-2), by MNC from MDS patients (using RT-PCR, zymography and Western blotting), as well as the effects of TNF-α on migration of MDS MNC across the reconstituted basement membrane Matrigel. We observed higher mean levels of TNF-α in media conditioned by mononuclear cells (MNC) obtained from 20 patients diagnosed with MDS (RAEB-T (4); RAEB (5), RA (10) and RARS (1)) in comparison to normal controls. We found that (i) MMP-9 is secreted by MDS MNC and this secretion is increased by TNF-α, (ii) MT1-MMP is expressed by the majority of MDS MNC and this expression is upregulated by TNF-α, and (iii) MMP-2 is detectable only in RAEB-T MNC samples and is activated by TNF-α. TNF-α also stimulated the migration of MDS MNC across Matrigel, which was inhibited by the inhibitor of MT1-MMP, epigallocatechin-3-gallate. Moreover, Enbrel (soluble TNF receptor fusion protein), which blocks TNF-α, also inhibited the expression of MT1-MMP and secretion of MMP-9 by MDS MNC as well as their migration across Matrigel. In addition, we observed increased activation of the latent form of MMP-2 in co-cultures of MDS MNC with bone marrow fibroblastic cells. We suggest that in MDS patients, TNF-α stimulates the expression of MMP-9 and MT1-MMP, inducing a highly proteolytic environment in bone marrow which could further increase processing and secretion of endogenous TNF-α. Therefore, we suggest that use of TNF-α inhibitors combined with MMP inhibitors could have therapeutic value in abrogating the progression of MDS.
SD-208, a Novel Transforming Growth Factor Beta Receptor I Kinase Inhibitor, Can Stimulate Hematopoiesis in Myelodysplastic Syndrome Progenitors.
Authors: Verma Amit , Navas Tony A. , Ying Jing , Nguyen Aaron N. , Pahanish Perry , Seetharam Mahesh , Kerr Irene , Collins Robert , Kim Mimi , Mohindru Mani , Higgins Linda S.
Published: April 2020

Transforming Growth Factor β (TGF-β) is a myelosuppressive cytokine that has been implicated in the ineffective hematopoiesis seen in myelodysplastic syndromes (MDS). Overactivation of TGF-β signaling in this disease was demonstrated immunohistochemically by significantly higher nuclear SMAD2 phosphorylation observed in 20 MDS bone marrows when compared with 7 non MDS anemic controls (P < 0.0001, 2 Tailed T Test, Image Pro Plus software). This data along with high levels of membrane-bound and plasma TGF-β observed in MDS patients in previous studies support the development of therapeutics targeting the TGF-β signaling pathways in this disease. SD-208 is a novel, potent and specific inhibitor of TGF-β Receptor I (TGFβ-RI) kinase. We demonstrate that SD-208 blocks the phosphorylation of SMAD2 in hematopoietic progenitors which are at the colony forming unit-erythroid (CFU-E) stage of differentiation. SD-208 also abrogates the G0/G1 cell cycle arrest induced by TGF-β in bone marrow progenitors. SD-208 treatment leads to reversal of the myelosuppressive effects of TGF-β on erythroid and myeloid colony formation from primary human CD34+ cells. Selectivity of SD-208 in inhibiting TGF-β-mediated effects on hematopoiesis was supported by similar results observed with siRNAs targeting SMAD2, a major component of the TGF-b signaling pathway. Finally, the efficacy of SD-208 in MDS was evaluated by treating bone marrow mononuclear cells from 15 patients with early low grade MDS. SD-208 treatment led to dose-dependent increases in erythroid and myeloid colonies after 14 days of in vitro culture. The effect was most notable in patients with high levels of activated SMAD-2, as assessed by immunohistochemical staining of bone marrow biopsies. Stimulation of hematopoiesis in MDS-derived marrow culture by SD-208 demonstrates a novel concept and potential therapeutic role for TGFβ-RI inhibition in this disease. Supported by VISN-17 grant, Harris Methodist Foundation Grant and ASCO YIA to AV
Predictive Factors for Response and Progression after Treatment with Rituximab in Previously Untreated Patients with Waldenstrom’s Macroglobulinemia (WM).
Authors: Anagnostopoulos Athanasios , Treon Steven P. , Zervas Konstantinos , Branagan Andrew , Gika Dimitra , Zomas Athanasios , Kyrtsonis Marie Christine , Vassou Amalia , Anagnostopoulos Nikolaos , Pangalis Gerasimos , Dimopoulos Meletios A.
Published: April 2020

Introduction: Rituximab is an active agent for the treatment of WM. However many patients do not respond to this agent and several others develop secondary resistance. The purpose of this study was to identify clinical and laboratory parameters which could predict a higher likelihood of benefit in previously untreated WM patients who received treatment with rituximab.

Patients and methods: We evaluated clinical and laboratory characteristics of 35 patients with WM who received primary treatment with single agent rituximab. We correlated these characteristics with response to treatment and time to progression.

Results: Between 8/98 and 12/03, 35 WM patients, median age 68 (range 39–90), 24 male, received primary treatment with extended rituximab as previously described (Dimopoulos et al, JCO 2002 and Treon et al, Ann Oncol 2005). Anemia (Hgb<10g/dL) was present in 55%, thrombocytopenia (PLT<100,000/mm3) in 31%, lymphadenopathy in 40%, splenomegaly, hyperviscosity and B-symptoms were present in 17% of the patients each. 43% of the patients had M-peak levels 4gr/dL, 37% had albumin <3.5g/dL and 56% had 2 microglobulin >3,5mg/dL. Fifteen patients (43%) achieved at least 50% reduction of M-peak and of tumor infiltrates at all involved sites. Patients with Hg10 g/dL and monoclonal kappa light chain had a significantly higher probability to respond [65% vs 22% (pvalue 0.01) and 50% vs 10 % (p value 0,037) respectively]. Patients with higher albumin and lower serum M-peak levels had also a higher frequency of response (p=0,15 and 0,09 respectively). After a median follow-up of 43 months, 19 patients have progressed, (5 responders and 14 non responders) with a median PFS of 23,6 months for the whole patient population and not reached for the responders (75% without progression at 26 months). In the univariate analysis the most important factor predicting longer PFS was hemoglobin 10gr/dL (median 57,8 vs 5,4 months). Other factors associated with longer PFS was albumin 3,5gr/L (median 25,5 vs 5,3 months), serum M-peak <4gr/L (48 vs 6,2), absence of hyperviscosity (23 vs 3,5) and presence of k light chain (25,4 vs 5,1).

Conclusion: Our analysis which included a large number of patients with long-term follow-up indicates that primary treatment with rituximab is associated with objective response in 43% of patients with WM and with a median PFS of 2 years. Patients most likely to benefit from rituximab include those without significant anemia, with k light chain, with lower levels of M-peak and with higher levels of albumin.
Increased Marrow CD57+ Cytotoxic T Cells Is a Powerful Prognostic Marker for Survival in Patients with Relapsed Multiple Myeloma (MM) Receiving Thalidomide.
Authors: Mileshkin Linda R. , Gambell Peter , Beshay Victoria , Hayakawa Yoshihiro , Smyth Mark , Trivett Melanie , Simmons Paul J. , Milner Alvin , Zeldis Jerome B. , Prince Miles
Published: April 2020

Background: In vitro studies indicate that thalidomide alters the marrow microenvironment and also has an immunoregulatory role. We assessed various laboratory and clinical parameters to examine potential prognostic markers and to assess for changes during thalidomide therapy.

Methods: 75 patients with relapsed/refractory MM were enrolled in a multi-centre phase 2 trial using thalidomide (Blood. 2003;102:69–77). Platelet-poor plasma (PPP) and marrow biopsy were obtained at baseline and 3 monthly and immunohistochemistry for CD34, VWF, mast cell tryptase and CD57 performed. Flow cytometry on marrow aspirates was used to define the CD57+ population (T cell subsets, NK, NKT markers used). Vascular endothelial growth factor (VEGF), basic-Fibroblast growth factor (bFGF), interleukin-6 (IL-6) and Hepatocyte growth factor (HGF) were measured in PPP. Objectives were to examine for effect on response rate (RR), progression-free (PFS) and overall survival (OS).

Results: Overall RR was 28% with 55% stable disease. Only VEGF predicted response-no responses seen in patients with a level of 0, compared to a RR of 34% in those with VEGF > 0 (p=0.015). Microvessel density (MVD) did not predict for response, PFS or OS. The median number of CD57+ cells at baseline was 3 per HPF (range: 0–27) and flow cytometry confirmed that CD57+ cells were predominantly cytotoxic T cells. CD57+ cells did not predict for response, however on univariate analysis elevated levels were the major predictor of better OS (p=0.003). Predictors for inferior OS were raised baseline levels of IL-6 (p=0.014), and HGF (p=0.016). Multivariate analysis for OS which incorporated clinical variables demonstrated age >65 yrs (p=0.009), raised LDH (p=0.008) and zero baseline CD57+ cells (p=0.011) as predictors of inferior OS. MVD and VEGF fell significantly in responding patients although CD57+ cells did not change.

Conclusion: Levels of VEGF and MVD decline in thalidomide responders. However, high baseline angiogenic activity was not necessary to obtain a response. Increased age and elevated LDH are important predictors of poorer OS, with elevated baseline levels of CD57+ cells being an independent predictor of superior outcome.
Juxtamembrane Domain Truncation Is Sufficient for Activation of the FIP1L1-PDGFRα and ETV6-PDGFRβ Fusion Kinases.
Authors: Stover Elizabeth H. , Chen Jing , Folens Cedric , Lee Benjamin H. , Mentens Nicole , Marynen Peter , Gilliland D. Gary , Cools Jan
Published: April 2020

Tyrosine kinase fusions such as BCR-ABL are common causes of myeloproliferative diseases and hematopoietic malignancies. Frequently, enforced dimerization by the fusion partner protein confers ligand-independent activation on these fusion kinases. FIP1L1-PDGFRα is a fusion kinase implicated in chronic eosinophilic leukemia and systemic mast cell disease. Recently, we showed that the partner protein FIP1L1 is not, in fact, required for PDGFRα activation in vitro or in vivo. We now demonstrate that truncation of the PDGFRα juxtamembrane (JM) domain is sufficient for kinase activation and subsequent transformation of Ba/F3 cells to growth factor independence, even in the absence of FIP1L1. In contrast, the presence of a complete JM domain is inhibitory to PDGFRα kinase activation. These findings are consistent with clinical data from patients with the FIP1L1-PDGFRα fusion, in which the breakpoints in PDGFRA are always tightly clustered within the JM domain. Furthermore, our results suggest that disruption of the JM domain, which has an autoinhibitory function in some type III receptor tyrosine kinases (RTKs), may contribute to activation of chimeric kinases. Notably, we observed a similar effect of JM domain truncation in a related type III RTK, PDGFRβ. Fusion of PDGFRB to various partner proteins, such as ETV6, can cause chronic myelomonocytic leukemia. However, we show that PDGFRβ kinase activation and signaling to downstream targets can be achieved in the absence of ETV6 via truncation of PDGFRB within the JM domain. These results suggest that disruption of an autoinhibitory JM domain may be an alternative, dimerization-independent mechanism of aberrant regulation of activated kinases involved in hematopoietic and other malignancies.