bluuebunny/crossref_metadata_2025 huggingface dataset:
Gains on Chromosome Band 9q34 Could Represent a Novel Independent Prognostic Marker in Multiple Myeloma Patients Receiving High-Dose Chemotherapy with Autologous Stem Cell Transplantation.
Authors: Liebisch Peter , Goldschmidt Hartmut , Tschajka Kathrin , Wendl Christiane , Benner Axel , Dohner Hartmut
Published: April 2020

Introduction: Genomic aberrations represent important prognostic markers in many hematological cancers. In multiple myeloma (MM), chromosome 13q deletion (13q-) has emerged as one of the most important outcome predictors and indicates a dismal prognosis. Other chromosomal abnormalities have been discussed as prognostic markers in this disease but came not out as independent variables when they were tested in a multivariate fashion. However, the complexity of genomic rearrangements and the clinical heterogeneity seen in malignant plasma cell disorders argue against 13q- as the sole genomic change of prognostic relevance.

Material and Methods: In a retrospective analysis, 109 patients (pts.) treated with one or two cycles of high-dose chemotherapy (HD-CTX) followed by autologous stem cell transplantation (ASCT) at a single center were analyzed by tri-color FISH and four DNA probes mapping to chromosome bands 1q21, 9q34, 11q25, and 13q14. The selection of DNA probes based upon previous data from a comprehensive molecular cytogenetic study that revealed a high incidence of chromosomal gains (+) and losses (−) involving these four loci. The Cox proportional hazards regression model was applied to correlate molecular cytogenetic markers with clinical data.

Results: The most frequent chromosomal abnormalities in the present series were +9q (49%), +1q (48%), +11q (47%), and 13q- (42%). The median follow-up time was 30 months (m) and the median event free survival (EFS) and overall survival (OS) time (calculated from first ASCT) of the entire cohort was 30 m and 71 m, respectively. There were 52 events (31 deaths). In a multivariable analysis including the four most frequent chromosomal abnormalities, +9q (hazard ratio 2.49, 95% CI 1.20-5.18; p=.01) and 13q- (2.34, 1.17–4.68; p=.02) were statistically significant risk factors for shorter EFS. The median EFS in pts. lacking both genomic changes was 3.6 years , while it was 2.5 years in pts. with either +9q or 13q- and only 1.5 years in pts. that exhibited both chromosomal abnormalities. Of note, when other potential prognostic factors ß2-microglobulin, albumin, number of chemotherapy cycles prior to first ASCT) were included in the multivariable analysis, +9q and 13q- remained independent risk factors for shorter EFS (p=.008 and p=.03, respectively). Due to the diversity of salvage treatment protocols applied after relapse, OS was not analyzed in this study.

Conclusions: +9q34 could represent a novel independent marker of adverse prognosis in MM pts. receiving HD-CTX with ASCT. The prognostic significance of +9q34 and other molecular cytogenetic aberrations is currently investigated within large multicenter trials on more homogenously treated cohorts of pts.
Inclusion of HOXA Genes in Genetic and Biological Networks Defining Human Acute T-Cell Leukemia.
Authors: Soulier Jean , Clappier Emmanuelle , Cayuela Jean-Michel , Regnault Armelle , Garcia-Peydro Marina , Dombret Herve , Baruchel Andre , Toribio Maria-Luisa , Sigaux Francois
Published: April 2020

We have identified a new recurrent chromosomal translocation, targeting the major homeobox gene cluster HOXA in human T-cell acute lymphoblastic leukemia (T-ALL). Four cases were characterized using a combination of FISH, Southern blot, breakpoint region sequencing, and a large scale expression analysis of a series of T-ALL. Specific RQ-PCR analysis of the HOXA1 to HOXA13 transcripts showed that the whole HOXA gene cluster expression was dramatically deregulated in the HOXA-rearranged cases, and also in the MLL and CALM-AF10-related T-ALL, strongly suggesting that HOXA genes are oncogenic in these types of leukemia. The HOXA-rearranged cases were included in a general portrait of T-ALL based on large scale expression analysis, showing that a new homogeneous T-ALL subgroup is defined by this chromosomal rearrangement. Moreover, patterns of gene expression associated to the distinct T-ALL oncogenic subgroups were compared with gene expression in normal human thymic sub-populations (11 purified sub-populations). Inappropriate use or perturbation of some specific molecular networks involved in thymic differentiation could be detected in the T-ALL cells. Also, we found that abnormal, frequently ectopic, expression of at least one developmental gene, including HOXA, TLX1/HOX11, TLX3/HOX11L2 and a few more, could be identified in most of the T-ALL cases. Our data strongly support the view that the abnormal expression of developmental genes, including the prototypical major homeobox genes HOXA in some cases, is critical in T-ALL oncogenesis.
Multiple Myeloma Oncogene 1 (MUM1)/Interferon Regulatory Factor 4 (IRF4) Upregulates Monokine Induced by Interferon-gamma (MIG) Gene Expression in B-Cell Malignancy.
Authors: Iida Shinsuke , Uranishi Miyuki , Sanda Takaomi , Ishida Takashi , Tajima Emi , Ito Masato , Komatsu Hirokazu , Inagaki Hiroshi , Ueda Ryuzo
Published: April 2020

MUM1(multiple myeloma oncogene 1)/IRF4(interferon regulatory factor 4) is a transcription regulatory factor that is activated as a result of t(6;14)(p25;q32) in multiple myeloma. MUM1 expression is seen in various B-cell lymphomas/leukemias and has been reported to predict an unfavorable outcome in some lymphoma subtypes including diffuse large B-cell lymphoma (DLBCL) and B-cell chronic lymphocytic leukemia (B-CLL). To elucidate its role in B-cell malignancies, we prepared stably MUM1-expressing Ba/F3 cells, which proliferated at a higher rate than the parental cells, and performed cDNA microarray analysis to identify genes whose expression is regulated by MUM1. We found that the expression of four genes including FK506-binding protein 3 (FKBP3), the Monokine induced by interferon-gamma (MIG), Fas apoptotic inhibitory molecule (Faim) and Zinc finger protein 94 was altered in the MUM1-expressing cells. We then focused on MIG since its expression was immediately upregulated by MUM1 in inducible MUM1 expressing system. In reporter assays, MUM1 activated the MIG promoter in cooperation with PU.1, and the interaction between MUM1 and the MIG promoter sequence was confirmed in chromatin immunoprecipitation assay. The expression of MIG was correlated with that of MUM1 in B-CLL cell lines, and its receptor CXCR3 was also coexpressed in B-CLL cell lines that were positive for MUM1. Interestingly, treatment with neutralizing antibodies against MIG and its receptor, CXCR3, partially inhibited the proliferation of two MUM1-expressing B-CLL cell lines. These results suggest that MUM1 plays certain roles in the progression of B-cell lymphomas/leukemias by regulating the expression of various genes including MIG.
Knockdown of the Transcription Factor PU.1 Causes Characteristic Transcriptional Changes in Hematopoietic Stem Cells Prior to Leukemic Transformation.
Authors: Steidl Ulrich , Rosenbauer Frank , Gu Xuesong , Owens Bronwyn , Klippel Steffen , Wagner Katharina , Aivado Manuel , Libermann Towia A. , Tenen Daniel G.
Published: April 2020

Knockdown of the expression of the transcription factor PU.1 in mice results in an initial preleukemic expansion of myeloid progenitors and subsequent transformation to an immature acute myeloid leukemia (AML). Recent reports suggest that functional inactivation of PU.1 might also play a role in human AML. However, the molecular mechanisms underlying the malignant transformation are unknown. Examining the bone marrow of leukemic PU.1 knockdown mice we found an expansion of lin−, c-kit+, Sca1+ hematopoietic stem cells (HSC) as compared to wildtype animals. This finding prompted us to examine the transcriptome of HSC of PU.1 knockdown mice in the initial phase to look for early transcriptional changes underlying the malignant transformation. After lineage-depletion and FACS sorting of lin−, c-kit+, Sca1+ HSC we performed linear amplification of RNA which was then hybridized with Affymetrix Mouse Genome 430 2.0 arrays covering ~39,000 transcripts. Unsupervised hierarchical cluster analysis clearly distinguished PU.1 knockdown and wildtype HSC, demonstrating that reduced PU.1 expression levels lead to characteristic transcriptional changes in the stem cell compartment preceding the malignant transformation. 55 transcripts were upregulated, and 143 transcripts were downregulated in PU.1 knockdown HSC (>1.5-fold, p<0.05), reflecting the role of PU.1 as a transcriptional activator. We corroborated differential expression of 16 genes by quantitative real-time RT-PCR. Among the downregulated genes were c-fes, Bruton’s tyrosine kinase, transcription factor EC, and GM-CSF receptor α, demonstrating that those in-vitro targets of PU.1 are also affected in stem cells in vivo. BAFF-R and TACI, two members of the tumor necrosis factor receptor superfamily, receptor tyrosine kinase TEK and toll-like receptor 4 were downregulated, whereas PDGF receptor was upregulated, suggesting disturbed signaling in PU.1 knockdown HSC. We are currently investigating the causal contribution of these deregulated genes to the leukemic phenotype. In summary, our data delineate a distinct transcriptional pattern that precedes the leukemic transformation in PU.1 knockdown HSC, and provide novel insight into PU.1-mediated molecular pathways in vivo.
Ki23819 (KRN383•HCl) Inhibits Kinase Activity of Wild Type and Mutant FLT3 Receptor Tyrosine Kinase In Vitro.
Authors: Komeno Yukiko , Kurokawa Mineo , Imai Yoichi , Takeshita Masataka , Matsumura Tomoko , Kubo Kazuo , Yoshino Tetsuya , Nishiyama Uichi , Kuwaki Tomoaki , Kubo Kinya , Osawa Tatsushi , Ogawa Seishi , Chiba Shigeru , Miwa Atsushi , Hirai Hisamaru
Published: April 2020

[Background] FLT3 is a class III receptor tyrosine kinase which is widely expressed on hematopoietic stem/progenitor cells. Two types of constitutively active FLT3 mutations have been reported to be expressed on a subset of leukemic cells; internal tandem duplications (ITD) and kinase domain mutations. The former are associated with poor prognosis in acute myeloid leukemia (AML) patients. Although several inhibitors targeting FLT3-ITD are tested in clinical trials, their cytotoxic effects are still unsatisfactory. Innate and acquired resistance is also a problem to be solved. [Purpose] To screen a novel potent FLT3 inhibitor and characterize its in vitro activity. [Materials and Methods] MOLM13 and MV4-11 cells, human leukemia cell lines expressing FLT3-ITD, were exposed to candidate compounds for 48 hours, and cytotoxic effect was assessed by colorimetric assay. Inhibitory effect on autophosphorylation was evaluated by immunoprecipitation and Western blotting. These effects were also tested in 32D cells engineered to express wild type FLT3 (FLT3-WT) or FLT3-ITD. FLT3-WT was activated with 50 ng/ml FLT3 ligand for 15 min. Proapoptotic effect was confirmed by flow cytometry with Annexin V staining. In vitro kinase assay was performed to demonstrate direct inhibition of tyrosine kinase activity of FLT3-ITD. Inhibitory effects on downstream signaling molecules, ERK and STAT5, were assessed by Western blotting. [Results] Among candidates for VEGFR inhibitors from a library, a quinoline-urea derivative Ki23819 (KRN383•HCl) was identified to specifically inhibit proliferation and induce apoptosis to MOLM13 and MV4-11 cells. Ki23819 inhibited proliferation of MV4-11 cells more effectively than SU11248, a precedent FLT3 inhibitor (IC50 <1 nM vs 3~10 nM). Similar results were obtained when MOLM13 cells were used. Ki23819 inhibited autophosphorylation of both ligand-activated FLT3-WT and FLT3-ITD (IC50 30 nM and 3 nM, respectively), and abrogated IL-3-independent proliferation of 32D cells expressing FLT3-ITD (IC50 3~10 nM). In vitro kinase assay demonstrated direct inhibition of kinase activity of FLT3-ITD (IC50 7.8 nM). This compound also inhibited ERK and STAT5 constitutively activated by FLT3-ITD. The IC50 for inhibition of phosphorylation in 32D FLT3-ITD cells was 3 nM for both proteins, which is equivalent to that for inhibition of FLT3-ITD autophosphorylation. [Conclusion] Ki23819 is a novel and potent candidate for antileukemic agents against FLT3-ITD positive AML. In vivo activity of KRN383, the free base of Ki23819, is also to be reported in this ASH meeting (Nishiyama et al.).
Effect of LCR 5′HS4 and 5′HS1 Deletions on β-Like Globin Gene Expression in β-Yac Transgenic Mice.
Authors: Harju Susanna , Fedosyuk Halyna , Peterson Kenneth R.
Published: April 2020

A 213 Kb human β-globin locus yeast artificial chromosome (β-YAC) was modified by homologous recombination to delete 2.9 Kb of cross-species conserved sequence similarity encompassing the LCR 5′HS4 (Δ5′HS4 β-YAC). Three transgenic mouse lines were established; each contained two intact copies of the β-globin locus as determined by long range restriction enzyme mapping (LRRM) and Southern blot hybridization analyses. Human ε-, γ- and β-globin, and mouse α- and ζ-globin mRNAs were measured by RNAse protection in hematopoietic tissues derived from staged embryos, fetuses and adult mice. No difference in the temporal pattern of globin transgene expression was observed between Δ5′HS4 β-YAC mice and wild-type β-YAC mice. In addition, quantitative per-copy human β-like globin mRNA levels were similar between Δ5′HS4 and wild-type β-YAC transgenic lines, although γ-globin gene expression was slightly increased in the fetal liver, while β-globin gene expression was slightly decreased in Δ5′HS4 β-YAC mice. These data are in contrast to data obtained from β-YAC mice containing a deletion of the 280 bp 5′HS4 core. In these mice, γ- and β-globin gene expression was significantly decreased during fetal definitive erythropoiesis and β-globin gene expression was decreased during adult definitive erythropoiesis. However, these data are consistent with the observation that deletion of the 5′HS core elements is more deleterious than large deletions of the 5′HSs. Together, the compiled deletion data supports the hypothesis that the LCR exists as a holocomplex in which the 5′HS cores form an active site and the flanking 5′HS regions constrain the holocomplex conformation. In this model, 5′HS core mutations are dominant negative, whereas larger deletions allow the LCR to fold into alternate holocomplex structures that function normally, albeit less efficiently.

To complete the study on the contribution of the individual 5′HSs to LCR function, a 0.8 Kb 5′HS1 fragment was deleted in the 213 Kb β-YAC by homologous recombination. Two ΔHS1 β-YAC transgenic lines have been established; four additional founders were recently identified. Of the two lines, one contains two intact copies of the globin locus; the other contains four deleted copies, one of which extends from the LCR through just 5′ to the β-globin gene. For both lines, ε-globin gene expression was markedly reduced, approximately 5–10 fold, during primitive erythropoiesis. Developmental expression profiles and levels of the γ- and β-globin genes (in the line that contains loci including the β-globin gene) were unaffected by deletion of 5′HS1. Breeding of the remaining four founders to obtain F1 and F2 progeny for similar structure/function studies is in progress. Decreased expression of the β-globin gene is the first phenotype ascribed to a 5′HS1 mutation, suggesting that this HS does indeed have a role in LCR function.
In Vivo and In Vitro Analysis of an Aγ-Globin Gene Silencer in Human β-Yac Transgenic Mice.
Authors: Harju Susanna , Peterson Kenneth R.
Published: April 2020

Autonomous silencing of gene expression is one mechanism operative in the control of human β-like globin gene switching and is best exemplified by the ε-globin gene. Experiments using variously truncated Aγ-globin genes linked to LCR sequences suggested that a region of the Aγ-globin gene between −730 to −378 relative to the mRNA CAP site may function as an adult stage-specific silencer element. A 5.4 Kb marked Aγ-globin gene (Aγm) inserted between LCR 5′HS1 and the ε-globin gene in a β-YAC (Aγm 5′ ε β-YAC) was silenced in transgenic mice during adult definitive erythropoiesis, even in the absence of an adult β-globin gene. In contrast, when a marked β-globin gene (βm) was inserted in this same location in another β-YAC (βm 5′ ε β-YAC), the βm-globin gene was expressed throughout ontogeny. From these data we concluded that: 1) any gene located near the LCR will be strongly expressed throughout ontogeny, unless some gene-specific silencing mechanism exists, 2) competition between the γ- and β-globin genes for interaction with the LCR is not the exclusive mechanism controlling γ- to β-globin gene switching, and 3) that the Aγm-globin gene was autonomously silenced. A -730 to -378 deletion of the Aγm-globin gene was introduced into the Aγm 5′ ε β-YAC via homologous recombination to produce a Δ1s Aγm 5′ ε β-YAC. This YAC was microinjected and six founders were obtained. Four transgenic lines were established carrying at least one full-length β-globin locus and two were established that lacked the adult β-globin gene. All founders containing an intact β-globin gene expressed the Δ1s Aγm-globin during adult erythropoiesis (45% – 122% relative to human β-globin expression). In one line examined in detail, the Δ1s Aγm-globin gene was expressed in the embryonic yolk sac, fetal liver, and adult blood. ε-globin gene expression was not detected in the embryonic yolk sac and expression of the normally located γ-globin genes was not observed at any developmental stage. β-globin gene expression was observed in the fetal liver and adult blood, although its expression was decreased. To further delineate the function of the Δ1s fragment, transient transfection assays to test silencer function and protein-DNA interaction assays were performed. Silencer activity of the352 bp Δ1s fragment was examined using a series of pGL2 luciferase reporter plasmids that were synthesized to include the Δ1s fragment; these were electroporated into various cells. Electrophoretic mobility shift assays (EMSAs) and DNAse I footprinting were employed to begin assessment of protein binding within the Δ1s fragment. A 50 bp DNA fragment spanning −713 to −664 of the Δ1s element was used in EMSAs; DNA binding activity was observed in K562 nuclear extracts. These preliminary data suggest that the −730 to −378 γ-globin gene silencer binds a repressor protein complex.
B Locus YAC Mice Carrying G Gene Promoter Mutations: Insights into the Mechanism of Action of the -175 HPFH Mutation.
Authors: Yu Man , Ye Xin , Han Hemei , Stafford Mary , Navas Patrick A. , Li Qiliang , Stamatoyannopoulos George
Published: April 2020

Non-deletional HPFHs are g gene promoter mutations, which are associated with HbF elevation in heterozygous adult individuals. To investigate the mechanism whereby these mutations result in g gene activation, the −198 (T→C) and −175 (T→C) mutations were introduced into the promoter of the Ag gene in the context of a 213 kb b-globin locus YAC. The mutated YAC DNA was then used to produce transgenic mice. Expression of the human g and b globin gene was examined by RNase protection assay in blood of the adult transgenic mice carrying either −198 or −175 mutations. The level of g gene expression ranged from 4.0 to 8.5% of the human b mRNA in the −198 mutant transgenic mice, and from 25 to 41% in the −175 mutant mice. In contrast, there was no detectable level of g gene expression in the blood of wild type transgenic mice. The ratios of g/b mRNA in the mutant transgenic mice correspond to the levels of Hb F in adult individuals carrying the corresponding mutations: Hb F in heterozygotes for HPFH −198 and −175 mutations ranges from 3.5 to 10% and from 36 to 41%, respectively. To delineate the mechanism of g gene reactivation by the HPFH mutations, the CACCC box in the g gene promoter was deleted in the context of the mutant YAC. In contrast to the −175 HPFH bYAC mice, in the doubly mutated −175 HPFH DAgCACCC bYAC mice, expression of the g gene in the adult blood of the transgenic mice was undetectable. These results provide strong evidence that an intact CACCC box is required for g gene activation in the −175 HPFH mutation. We conclude that YAC mice can faithfully reproduce the phenotypes of nondeletional HPFH mutations and be used for the investigation of the mechanisms by which these mutations activate the g gene.
Poor Outcome of Allogeneic Bone Marrow Transplantation Recipients Requiring Mechanical Ventilation: Should We Restrict Intensive Care Support?.
Authors: Aubron Cécile , Azoulay Elie , Blot François , Mira Jean-Paul , Thiery Guillaume , Raynard Bruno , Buzyn Agnes , Socié Gérard , Arnaud Philippe , Pene Frédéric
Published: April 2020

In the recent years, a better selection of patients and early management of organ dysfunction allowed an improved survival of critically ill hematological patients. However allogeneic bone marrow transplantation (BMT) recipients admitted to the ICU with severe organ dysfunction still have poor survival rates. We performed a large retrospective multicenter study to analyse the features and short-term and long-term outcomes of allogeneic BMT recipients admitted to the ICU. Between 1997 and 2003, 220 allogeneic BMT recipients were referred to 3 ICUs. Patients were classified in three groups, on the basis of time since BMT : group 1 (< Day 30) n= 73, group 2 (Day 31 to day 100) n= 59, group 3 (> day 100) n=88. The ICU overall mortality rate was 51% (112/220), 56% (41/73) in the group 1, 54% (32/59) in the group 2 and 44% (39/88) in the group 3. Twenty percent of patients (45/220) were alive at 6 months after ICU admission and 14% (31/220) were alive one year after. None of the pre-admission characteristics (age, underlying disease and disease status at the time of transplantation, type of transplant) was associated with prognosis in the ICU. Mortality rate was highly correlated with the number of organ dysfunctions as measured by the Logistic Organ Dysfunction (LOD) score at admission (LOD score < 5 : 30%, LOD score = 6 to 10 : 56%, LOD score ≥ 11 : 96%). Of the 119 patients who required invasive mechanical ventilation, 83% died (98/118). Among the ventilated survivors, 11% (13/118) and 6% (7 /118) remained alive at 6 months and one year respectively. In a multivariate logistic regression analysis, the requirement of invasive mechanical ventilation (OR 19.5, CI 95% 8.8–45.3, p<0.0001) and hepatic dysfunction with elevation of serum bilirubin >68 μmol/L (OR 4.7, CI 95% 1.9–11.4, p=0.0006) were independently associated with mortality. Although the requirement of invasive mechanical ventilation remains desperately associated with a high mortality rate, some allogeneic BMT recipients may have a good long-term outcome. Assessment of other organ failures, and particularly hepatic dysfunction, may lead to early identification of patients with very high probability of death.
Differential Requirements for Survivin in Hematopoietic Cell Development.
Authors: Xu Yanfei , Gurbuxani Sandeep , Keerthivasan Ganesan , Wickrema Amittha , Crispino John D.
Published: April 2020

The development of the complete repertoire of blood cells from a common progenitor, the hematopoietic stem cell, is a tightly controlled process that is regulated, in part, by the activity of lineage specific transcription factors. Despite our knowledge of these factors, the mechanisms that regulate the formation and growth of distinct, but closely related lineages, such as erythroid cells and megakaryocytes, remain largely uncharacterized. Here we show that Survivin, a member of the inhibitor of apoptosis (IAP) family that also plays an essential role in cytokinesis, is differentially expressed during erythroid versus megakaryocyte development. Erythroid cells express Survivin throughout their maturation, up to the terminal stage of differentiation (orthochromatic), even after the cells exit the cell cycle. This is surprising because Survivin is generally expressed in a cell cycle dependent manner and not thought to be expressed in terminally differentiated cells. In contrast, purified murine megakaryocytes express nearly 5-fold lower levels of Survivin mRNA compared to erythroid cells. To investigate whether Survivin is involved in the differentiation and/or survival of hematopoietic progenitors, we infected primary mouse bone marrow cells with retroviruses harboring either the human Survivin cDNA or a mouse Survivin shRNA, and then induced erythroid and megakaryocyte differentiation in both liquid culture and colony-forming assays. These studies revealed that overexpression of Survivin promoted the terminal differentiation of red blood cells, while its reduction, by RNA interference, inhibited their differentiation. In contrast, downregulation of Survivin facilitated the expansion of megakaryocytes, and its overexpression antagonized megakaryocyte formation. In addition, consistent with a role for survivin in erythropoiesis, downregulation of Survivin expression in MEL cells led to a block in terminal differentiation. Finally, since caspase activity is known to be required for erythroid maturation, we investigated whether survivin associated with cleaved caspase-3 in erythroid cells. Immunofluorescence revealed that Survivin and cleaved caspase-3 co-localized to discrete foci within the cytoplasm of erythroid cells at the orthochromatic stage of development. Based on these findings, we hypothesize that Survivin cooperates with cleaved caspase-3 in terminal maturation of red blood cells. Together, our findings demonstrate that Survivin plays multiple, distinct roles in hematopoiesis.
Rituximab Plus Hypercvad (R-HCVAD) Alternating with Rituximab Plus High-Dose Methotrexate-Cytarabine (R-M/A) in Untreated Mantle Cell Lymphoma (MCL): Prolonged Follow-Up Confirms High Rates of Failure-Free Survival (FFS) and Overall Survival (OS).
Authors: Romaguera Jorge Enrique , Fayad Luis , Rodriguez Maria A. , Hagemeister Fredrick B. , Pro Barbara , McLaughlin Peter , Younes Anas , Samaniego Felipe , Goy Andre , Sarris Andreas H. , Dang Nam H. , Medeiros L. Jeffrey , Katz Ruth , Gagneja Harish , Samuels Barry I. , Cabanillas Fernando
Published: April 2020

Introduction: Aggressive MCL has a poor prognosis with a 21-40% complete remission (CR) after CHOP and a duration of response of only 10-16 months. More intense therapy could improve these statistics. Rituximab is effective in MCL and has minimal toxicity.

Methods: A prospective phase II trial of R-HCVAD (considered to be one cycle) alternating every 21 days with R- M/A (considered to be another cycle) as described earlier (Ann Oncol. 13, suppl 2, 2002 #24). Prophylaxis with mesna, calcium leucovorin, prednisone eyedrops, G-CSF, antibacterial, antifungal, and antiviral therapy. CBC with differential and platelet counts X 2-3/week. Re-staging every 2 cycles including upper and lower endoscopies. Patients in complete remission (CR) after 6 courses of a planned 6-8 cycles were not offered consolidation with stem cell transplant. Post-treatment evaluation was performed every 3 months for 1 yr, every 4 months for 2 yrs, every 6 months for 2 years, then annually.

Results: Of 100 patients registered, one was ineligible and two decided to not receive the treatment after registration, leaving 97 evaluable for analysis of response, survival and toxicity. An analysis of response after the first 6 cycles shows an 87% CR/CRu rate. With a median follow up of 40 months, the 3-year FFS and overall survival (OS) were 67% and 81%, respectively. Adverse factors for FFS were:

Grade 4 hematologic toxicity was significant. Five patients died during treatment of sepsis (3), pulmonary hemorrhage (1), and unknown cause (1). Four patients developed myelodysplasia/acute myelogenous leukemia after treatment and while in CR and three have died, for a total of 8 deaths in the study (8%).

Conclusion: R-HCVAD alternating with R-M/A without stem cell transplant is an effective regimen for treatment of aggressive untreated MCL, specially for patients ≤ 65 years old. Toxicity is as expected for an intense regimen. This encouraging data warrants continued follow-up and comparison with existing/new therapies in future trials.
Anti-Tumor Activity of Single-Agent CCI-779 for Relapsed Mantle Cell Lymphoma: A Phase II Trial in the North Central Cancer Treatment Group.
Authors: Witzig Thomas , Geyer Susan , Ghobrial Irene , Inwards David , Fonseca Rafael , Kaufmann Scott , Kurtin Paul , Colgan Joseph , Morice William , Alberts Steven , Salim Muhammed , Rowland Kendrith , Dakhil Shaker , Morton Roscoe , Stella Philip
Published: April 2020

Purpose: Mantle cell lymphoma (MCL) is characterized by a t(11;14) resulting in overexpression of cyclin D1, a member of the phosphatidylinosital 3 kinase (PI3K) pathway. This study tested whether CCI-779, which inhibits the PI3K pathway at the level of the mammalian target of rapamycin (mTOR) could produce tumor responses in patients (pts) with MCL.

Patients and Methods: Eligible pts had biopsy-proven, cyclin D1 positive MCL and had relapsed or were refractory to therapy. Pts received CCI-779 250 mg IV every week as a single agent. Pts were re-staged after 1 cycle (4 doses) and every 3 cycles thereafter. Pts with a tumor response after 6 cycles were eligible to continue drug for a total of 12 cycles or 2 cycles after complete remission (CR) and then were observed.

Results: Thirty-five pts were enrolled and evaluable for toxicity; 1 patient had MCL by histology but was cyclin D1 negative and ineligible for efficacy evaluation. The median age was 70 years (range, 38–89), 91% were stage 4, and 69% had ≥ 2 extranodal sites. Pts had received a median of 3 prior therapies (range, 1–11) and 54% were refractory to their last treatment. The overall response rate was 38% (13/34) with 1 CR (3%) and 12 PRs (35%), surpassing the pre-defined criteria for a promising agent. Responses tended to occur rapidly with median time to response of 1 month (range, 1–8). To date, 26 patients have progressed, with a median time-to-progression of 6.8 months (95% CI: 3.8 – 9.7). Median duration of response for the 13 responders was 5.7 months (95% CI: 5.2 – 13.2). Overall, 32 out of 35 patients who received treatment had grade 3 or 4 toxicity. The most common toxicities were hematologic with grade 3 (n=24) or grade 4 (n=4). Thrombocytopenia was the most frequent grade 3/4 toxicity (n=25) and the largest cause of dose-reductions, although counts typically recovered within one week. Only 4 patients could tolerate sustained 250 mg per week throughout their treatment (including one who went on to alternate treatment after 1 cycle) and the median dose/month was 175 mg.

Conclusions: Single-agent CCI-779 has substantial anti-tumor activity in relapsed MCL. This study demonstrates that agents, which selectively target cellular pathways dysregulated in MCL cells can produce therapeutic benefit. The high response rate warrants further studies of this agent in MCL, but the high incidence of hematologic toxicity suggests that a lower dose should be explored. CCI-779 at 25mg is currently being evaluated in MCL through an NCCTG trial
Secondary Amenorrhoea after Hodgkin’s Lymphoma Is Influenced by Age at Treatment, Stage of Disease, Chemotherapy Regimen, and the Use of Oral Contraceptives during Therapy: A Report from the German Hodgkin Lymphoma Study Group (GHSG).
Authors: Behringer Karolin , Breuer Kai , Reineke Thomas , May Michael , Nogova Lucia , Klimm Beate , Diehl Volker , Engert Andreas
Published: April 2020

Purpose: Long term survivors of successfully treated Hodgkin’s disease (HD) are at risk for late complications. Among these, infertility for female patients is of major importance affecting many young women. The subject of this analysis is to evaluate the menstrual status in young women after HD therapy.

Patients and methods: From 1994–1998, the German Hodgkin Lymphoma Study Group (GHSG) conducted clinical trials for early, intermediate and advanced-stage HD (HD7-HD9) involving a total of 3186 patients. A questionnaire was used to evaluate the menstrual status of young female patients after therapy. The following factors were assessed by multivariate analysis for their influence on amenorrhoea: age, treatment, stage, and the use of oral contraceptives during chemotherapy.

Results: The data presented were last updated in May 2004. A total of 405 women aged <40 years answered the questionnaire referring the menstrual status after HD therapy. For most women (89.6%), menstruation before the beginning of therapy was regular. After a median follow-up of 3.2 years, 51.6% of the women receiving 8 cycles of dose-escalated BEACOPP had continuous amenorrhoea. Amenorrhoea was significantly more frequent in women receiving 8 cycles of dose-escalated BEACOPP compared to women treated with ABVD alone, COPP/ABVD or standard BEACOPP (p=0.0066). Moreover, amenorrhoea after therapy was most pronounced in women with advanced-stage HD (p<0.0001), in those older than 30 years at treatment (p= 0.0065), and in women who did not take oral contraceptives during chemotherapy (p=0.0002).

Conclusion: Most women who are treated for advanced-stage HD experience amenorrhoea after therapy. Amenorrhoea is significantly more frequent in women with advanced-stage HD receiving 8 cycles of dose-escalated BEACOPP and in women older than 30 years at treatment. Furthermore, the present data suggest a possible protection of oral contraceptives against amenorrhoea.
Inhibition of Retinoic Acid Signaling by the cdx-hox Pathway Is Essential for Blood Cell Formation during Embryogenesis.
Authors: Davidson Alan J. , Wang Yuan , Daley George Q. , Zon Leonard I.
Published: April 2020

The zebrafish mutant kugelig (kgg) is caused by a defect in the caudal-related homeobox gene cdx4 and has a deficit in both ‘primitive’ and ‘definitive’ hematopoietic stem cells. The embryonic anemia in the mutants can be rescued by overexpressing hox genes such as hoxb7a and hoxa9a, but not hoxb8a. This suggests that specific hox genes are required to make mesoderm competent to form blood. To further explore this, we undertook a microarray analysis to identify differentially expressed genes in kgg mutants and wild-type embryos. We found that raldh2, an enzyme required for retinoic acid (RA) production, is overexpressed in kgg mutants during the early stages of blood formation. This data led us to hypothesize that RA may act to suppress blood formation and that the cdx-hox pathway functions to limit RA production, thereby permitting blood formation to occur. To test this, we treated wild-type zebrafish embryos with RA and found that they became severely anemic. Treating kgg embryos with DEAB, a chemical that blocks raldh2 activity, restored hematopoiesis in kgg mutants. Expression of hoxa9a was not rescued in these treated embryos, indicating that RA acts downstream of the hox genes. DEAB also induced an expansion of erythroid cells in wild-type embryos, thus supporting the notion that the levels of RA during development are a critical determinant for blood formation. By performing a time-course rescue experiment, we determined that DEAB is effective when scl + hematopoietic progenitors are first formed from mesoderm, suggesting that RA acts upstream of the blood-inducer scl. In support of this, SCL overexpression rescues GATA-1 expression in embryos treated with RA. We next looked at the effects of DEAB and RA on the formation of mouse hematopoietic progenitors arising from ES cell-derived embryoid bodies (EBs). Addition of DEAB to EBs between days 2 to 3 of development resulted in a 5–8 fold increase in ‘primitive’ erythroid colonies (CFU-Ep), analogous to our results in zebrafish embryos. In contrast, RA treatment caused a general inhibition in the growth of all colony types. Taken together, these results suggest a new model in which suppression of RA by the cdx-hox pathway is necessary for yolk sac hematopoiesis to occur. This model provides an explanation for how hox genes control the spatiotemporal formation of hematopoietic tissue during organogenesis and may shed new light on the pathogenesis of leukemias involving translocations of the cdx , hox, and retinoic acid receptors.
CD4+/bcl-6+ Peripheral T-Cell Lymphoma with Follicular Involvement: A Distinct Type of Nodal Peripheral T-Cell Lymphoma with Multiple T-Cell Clones.
Authors: Munster-Ikonomou Ida , Tierens Anne , Troen Gunhild , Aamodt Hege , Heim Sverre , Lauritzsen Grete , Delabie Jan
Published: April 2020

We studied six cases of a novel type of nodal peripheral T-cell lymphoma. Three cases with this disease were recently described by de Leval et al. (de Leval L, Savilo E, Longtine J, et al. Peripheral T-cell lymphoma with follicular involvement and a CD4+/bcl-6+ phenotype. Am J Surg Pathol2001;25:395–400). The entity was named peripheral T-cell lymphoma with follicular involvement because of its distinctive histological features. We report an additional six well-characterized cases and describe the molecular and cytogenetic findings. The neoplastic T-cells of this lymphoma type express CD4 and Bcl-6, and home to the B-lymphoid follicles. This suggests an origin of the lymphoma from an as yet poorly characterized subset of Bcl-6 expressing intra-follicular T-helper cells. Of interest, the cytogenetic data and/or the study of T-cell receptor gamma gene rearrangements revealed more than one clone in each case. Cytogenetics further revealed complex karyotypes without recurrent chromosomal aberrations. We also studied the presence of somatic mutations in the 5′ untranslated region of the BCL-6 gene in four of the cases but no somatic hypermutation was detected. Clinically, the cases presented with widespread lymph node involvement at diagnosis and multiple relapses occurred after treatment. All patients received a CHOP-based chemotherapy regimen, later followed by high dose chemotherapy with stem cell rescue in five patients. One patient died with lymphoma and hemophagocytic syndrome 24 months after diagnosis, one patient is alive with disease after 27 months from diagnosis, whereas the other four patients are in complete remission 12 to 124 months after diagnosis. In conclusion, we confirm that peripheral T-cell lymphoma with follicular involvement is a distinct lymphoma type and we show that the lymphoma is oligo-clonal. The clinical findings are those of an intermediately aggressive lymphoma type. Although minimal lymph node infiltration with lymphoma cells at diagnosis and oligo-clonality is also characteristic of angio-immunoblastic T-cell lymphoma, we believe that peripheral T-cell lymphoma with follicular involvement is a distinct T-cell lymphoma type. In contrast to angio-immunoblastic T-cell lymphoma it is characterized by the typical infiltration of lymphoma cells in B-lymphoid follicles, coagulation necrosis, the absence of proliferation of high endothelial venules and follicular dendritic cells in T-cell areas, as well as the absence of EBV infection. It is likely that T-cell lymphoma with follicular involvement arises from Bcl-6+ intra-follicular T-cells. No recurrent genetic defects have been identified but the oligo-clonal nature of the lymphoma is intriguing. The latter suggests that the triggering oncogenic factors are external, such as infection.
Rituximab, Bevacizumab (Avastin) and CHOP (RA-CHOP) for Patients with Diffuse Large B-Cell Lymphoma (DLBCL).
Authors: Ganjoo Kristen N. , Gordon Leo , Robertson Michael J. , Horning Sandra J.
Published: April 2020

Introduction

Bevacizumab is a humanized monoclonal antibody directed against vascular endothelial growth factor (VEGF). Non-Hodgkin’s lymphoma (NHL) patients with a high serum VEGF levels have an inferior overall survival compared to patients with low VEGF levels. Rituximab plus CHOP (R-CHOP) prolongs time to treatment failure and survival in patients with DLBCL. But further improvement is needed, particularly with patients with high-intermediate and high risk international index disease. This study was designed to assess the feasibility of the combination of bevacizumab plus R-CHOP (RA-CHOP).

Method

Bevacizumab was administered at 15 mg/kg IV on day 1 of each cycle over 30–90 minutes. R-CHOP was administered on day 2 of cycle 1 and on day 1 of subsequent cycles. Patients were monitored for cardiac function with echocardiogram and for proteinuria. Serum levels of bevacizumab and rituximab were measured to assess pharmacokinetics. Serum and urine samples were also analyzed for circulating VEGF. Tumor samples were stained for VEGF by immunohistochemistry.

Results

Twelve patients (10 males, and 2 females) with newly-diagnosed DLBCL were enrolled. The median age was 48 years (range 29–63). Three patients had stage II primary mediastinal, 2 stage III, and 7 stage IV disease. ECOG performance status ranged from 0–1. Nine patients had an elevated LDH with a median international index score of 2 (range 0–3). Nine patients had an international index score of 2–3. A total of 80 cycles (range 2–8; median 7) of RA-CHOP was administered. Bevacizumab was held in 5 cycles in 3 patients-due to 1) elevated liver enzymes in the setting of positive hepatitis C serology, 2) transient proteinuria, and 3) DVT associated with central line. The following grade 3 toxicities were observed: 4 febrile neutropenia with infection related to central line, 2 DVT (both central line associated), 1 elevated liver enzymes, and 2 transient hypertension. Grade 4 toxicities included 3 absolute neutropenia with 2 febrile neutropenia, and 1 HSV esophagitis. Two patients are too early to assess response. Best response included CR in 3, PR in 4 (2 with normal PET scans), SD in 1. One patient progressed and another achieved a PR after 4 cycles but further treatment was not given due to severe depression.

Conclusion

In this patient population, treatment with RA-CHOP did not result in any grade 3 or 4 proteinuria, heart failure or bleeding. Hypertension was transient and thrombosis was associated with central line placement. Correlative studies and pharmacokinetics are being analyzed and will be available for presentation.
Survivin Expression, Regulation and Biological Role in Multiple Myeloma.
Authors: Romagnoli Mathilde , Bataille Régis , Barillé-Nion Sophie
Published: April 2020

Multiple myeloma (MM) is characterized by the accumulation within the bone marrow of malignant plasma cells with an enhanced survival capacity. Myeloma cells often develop drug resistance leading to treatment failure in patients. Survivin is a member of the inhibitor of apoptosis (IAP) gene family that has been implicated in both cell viability and regulation of mitosis in cancer cells. In this study, we have evaluated survivin expression and its biological involvement in viability, proliferation, cell cycle and drug resistance in myeloma cells. First by western blotting we detected survivin expression in 17 human myeloma cell lines (HMCL) from moderate level in the HMCL XG6 to strong level in the HMCL U266. Survivin was also detectable in primary myeloma cells purified from blood or bone marrow samples of 20 patients in contrast to purified B lymphocytes from tonsil samples or autologous EBV infected B lymphocytes. Survivin expression peaked at G2/M phase as obtained by drug-induced cell-cycle arrest. Second, we demonstrated that both major myeloma growth factors, IL-6 and IGF-1, induced upregulation of survivin expression through JAK/STAT and PI3K/AKT signalling pathways. In order to elucidate survivin role in myeloma cells, we established XG6 stable transfectants overexpressing survivin and extinguished survivin expression by siRNA in U266. Preliminary data suggest that survivin may participate in spontaneous cell death regulation, cell proliferation and drug sensitivity in those HMCL. In summary, our findings tend to show that survivin may be playing an important role in the pathogenesis of MM. A more defined understanding of survivin biology should enhance the rational development of drugs to inhibit its function in myeloma cells.
The Bone Marrow Microenvironment Influences the Differential Chemokine Receptor Expression of Normal and Neoplastic Plasma Cells.
Authors: Davies Faith E. , Al Rayes Mona H. , Child J. Anthony , Morgan Gareth J. , Rawstron Andrew C.
Published: April 2020

The expression of cytokines and chemokines are under control of several factors, including the bone marrow (BM) microenvironment. The aim of this work was to study the chemokine receptor expression on normal and neoplastic PCs and to investigate the relationship between the BM microenvironment and plasma cell behaviour. The study included 20 patients with reactive disorders or normal BM, 20 individuals with MGUS (monoclonal gammopathy of undetermined significance) and 19 patients with multiple myeloma at presentation. A large panel of chemokine receptor-specific antibodies directed against CCR1, CCR2, CCR3, CCR5, CCR6, CXCR1, CXCR2, CXCR3, CXCR4 and CXCR5 was characterized employing a four-colour flow cytometry approach. We demonstrate that normal and myeloma PCs have a specific chemokine receptor profile expressing 7/10 of the receptors studied. CCR2, CCR6, and CXCR1 showed decreased expression on myeloma PCs in comparison to normal PCs (average 3.3, 1.5 and 1.8-fold difference in expression level respectively). In contrast CXCR4 was upregulated on myeloma PCs on average 2.2-fold in comparison to normal PCs. There was no significant difference in expression between normal (CD19+) and neoplastic (CD19−) PCs from the same bone marrow environment in MGUS patients with respect to CCR6, CXCR1 and CXCR4. In contrast there was a significant difference in expression of CCR2 between CD19+ and CD19− PCs from the same BM (P=0.002). The level of expression on CD19− PCs was on average 1.6-fold lower than on their CD19+ counterparts (range 1.1 6.8-fold lower). In conclusion these data demonstrate that normal and myeloma PCs have a specific chemokine receptor profile. Myeloma PCs have a reduced level of some chemokine receptors compared to normal PCs which may account for their abnormal localization within the BM. Differences in expression of CXCR1, CXCR4, and CCR6 are not specific to the neoplastic process, as both normal and neoplastic plasma cells from the same marrow in MGUS patients show corresponding levels of expression. It is probable that feedback loops between neoplastic plasma cells and bone marrow stroma also influence normal plasma cell expression of these chemokine receptors. However, differences in CCR2 expression are not influenced by the marrow microenvironment, therefore downregulation of CCR2 expression is either a function of the neoplastic process or of the stage of differentiation of the originating neoplastic cell. Interfering with chemokines and their receptors which are related to the malignant transformation, particularly CCR2, may prove useful as adjunct to chemotherapy approaches.