bluuebunny/crossref_metadata_2025 huggingface dataset:
MAP Kinase Activation Is Essential for the Development of Acute Leukemia by MLL Fusion Protein and FLT3 Tyrosine Kinase Mutation.
Authors: Ono Ryoichi , Kumgai Hidetoshi , Nakajima Hideaki , Tonozuka Yukio , Hishiya Ai , Taki Tomohiko , Hayashi Yasuhide , Kitamura Toshio , Nosaka Tetsuya
Published: October 2019

MLL (mixed lineage leukemia)-fusion-mediated acute leukemia in infants has frequently been found to have FLT3 overexpression or tyrosine kinase domain (TKD) mutation. We have recently developed both in vitro and in vivo leukemogenesis models where MLL fusion proteins cooperate with another FLT3 mutant, internal tandem duplication (ITD). However, little has been clear about molecular mechanism of the cooperativity between MLL fusion protein and FLT3 mutants, not only FLT3-ITD but also FLT3-TKD. The present study demonstrates that MLL-SEPT6 fusion protein associated with infantile acute leukemia cooperates in vitro with FLT3-TKD mainly through activated MAP kinase pathway, while MLL-SEPT6 cooperates with FLT3-ITD mainly through activated STAT5 pathway. We first found that the interleukin (IL) -3 dependent murine hematopoietic cell line immortalized by MLL-SEPT6, named HF6, was transformed to grow without IL-3 by forced expression of FLT3 mutants which activated MAP kinase and STAT5, as shown in IL-3 dependent murine pro-B Ba/F3 cells. A dominant negative mutant of STAT5A suppressed the proliferation of the HF6 cells transformed by FLT3-ITD more effectively than that by FLT3-TKD, similarly to the transformed Ba/F3 cells. However, unlike the transformed Ba/F3 cells, the proliferation of transformed HF6 cells was suppressed with an MEK inhibitor more effectively in the HF6 cells transformed by FLT3-TKD than by FLT3-ITD. These results suggested that, in the transformation of HF6 cells, MAP kinase activation is more critical for FLT3-TKD than STAT5, while STAT5 activation is more critical for FLT3-ITD than MAP kinase. Furthermore, HF6 cells became IL-3 independent by direct activation of Raf-MAP kinase, while Ba/F3 cells did not. In contrast, a constitutively active mutant of STAT5 enabled, not HF6, but Ba/F3 cells to grow without IL-3, thus suggesting the essential role of activation of the Raf-MAP kinase cascade in the growth of the cells expressing MLL fusion protein. We next examined the oncogenic potential of MLL-SEPT6 and either of the FLT3 mutants by leukemogenesis assays in vivo using bone marrow transplantation. Interestingly, FLT3-TKD cooperated with MLL-SEPT6 in vivo to induce acute leukemia in mice rapidly (26±5.5 days), similarly to FLT3-ITD (27±5.1 days), although the individual oncogenic potential of FLT3-TKD leading to T-cell lymphoma (119±11 days), was much weaker than that of FLT3-ITD leading to myeloproliferative disease (56±16 days). Taken together, these results suggest that MLL fusion protein can induce human acute leukemia in concert with MAP kinase activation through secondary genetic events including FLT3-TKD mutation or other mechanisms which activate MAP kinase.
Cyclin D2 Dysregulation by Chromosomal Translocations to TCR Loci in T-Cell Acute Lymphoblastic Leukemia (T-ALL).
Authors: Clappier Emmanuelle , Cuccuini Wendy , Cayuela Jean-Michel , Vecchione Danielle , Baruchel Andre , Dombret Herve , Sigaux Francois , Soulier Jean
Published: October 2019

D-type cyclins are key regulators of progression through G1 phase of the cell cycle. Strong expression of at least one of the three D cyclins is common in human cancers. However, while the cyclin D1 and D3 genes (CCND1 and CCND3) are recurrently involved in genomic rearrangements, especially in mantle-cell lymphoma and multiple myeloma, no clear involvement of the cyclin D2 gene (CCND2) has been reported to date in human malignancies. In T-cell acute lymphoblastic leukemia (T-ALL), the T-cell receptor genes TCRA/D and TCRB are frequently involved in chromosomal rearrangements. In order to identify new genomic rearrangements and oncogenes in human T-ALL, we performed an interphasic FISH screening of T-ALL cases using TCR flanking probes. Using this approach, we identified two new chromosomal translocations: t(7;12)(q34;p13) and t(12;14)(p13;q11), involving the TCRB and TCRA/D loci, respectively. Molecular analysis of the breakpoint sequences demonstrated the involvement of the CCND2 locus at 12p13. Expression analysis using RQ-PCR and immunoblotting demonstrated dramatic cyclin D2 overexpression in the translocated cases (n=3) compared to other T-ALLs (total, n=86), whereas other genes located near the translocation breakpoints were not deregulated on microarray analysis. To further evaluate expression in T-ALL with respect to normal T-cell differentiation, we analyzed CCND2 expression in purified subpopulations from normal human thymus. CCND2 levels were downregulated through progression from the early stages of normal human T-cell differentiation and transition to beta-selection. In the most immature T-ALLs, a moderate CCND2 expression was observed, consistent with their differentiation stage, while low expression was found in other T-ALL. By contrast, the massive and sustained expression in the CCND2-rearranged T-ALL cases strongly suggested an oncogenic role due to the TCR translocation. T-ALL oncogenesis is a multi-step process; we here found that the TCR-CCND2 translocations were associated with other oncogene expression (TAL1, HOXA, or TLX3/HOX11L2), NOTCH1 activating mutations, and/or CDKN2A/p16/ARF deletion, showing that cyclin D2 dysregulation could contribute to multi-event oncogenesis in various T-ALL groups. In conclusion, this report is the first clear evidence of a direct involvement of cyclin D2 in human cancer due to recurrent somatic genetic alterations. This reinforces the view that the strong expression of cyclin D2 which is detected in various types of cancer can contribute to oncogenesis, and points to cyclin D2 as a potential target for therapy in these tumors.
Imatinib-Resistant Activation of β-Catenin by BCR-ABL in a Murine Model of Chronic Myelogenous Leukemia Stem Cells.
Authors: Minami Yosuke , Stuart Scott , Ikawa Tomokatsu , Banno Asoka , Hunton Irina , Willert Karl , Murre Cornelis , Jamieson Catriona , Wang Jean Y.J.
Published: October 2019

[INTRODUCTION] Chronic myelogenous leukemia (CML) is effectively treated with imatinib, a small molecule inhibitor of the BCR-ABL tyrosine kinase that is expressed in the hematopoietic compartment including stem and progenitor cells in CML patients. While imatinib induces disease remission, it does not eradicate BCR-ABL-positive stem cells. Recently, granulocyte-macrophage progenitors (GMP) with nuclear β-catenin and an aberrant potential for self-renewal were detected in CML blast crisis (Jamieson et al, NEJM, 2004). We have established a murine model that generates BCR-ABL-positive GMP and a transplantable CML. In this model, BCR-ABL activates β-catenin through an imatinib-resistant pathway.

[METHODS] Retroviral mediated gene transfer was employed to express BCR-ABL (p210) and/or GFP in a pluripotent murine hematopoietic progenitor cell line (mHPC) (Ikawa et al, Immunity, 2004). Infected cells were isolated using GFP-sorting and characterized ex vivo for leukemogenic phenotypes and gene expression profiles. The GFP-sorted cells were transplanted into lethally irradiated congenic mice. The distribution of GFP-positive cells in the hematopoietic compartments was determined by FACS.

[RESULTS] BCR-ABL expression induced leukemogenic traits in mHPC, i.e., stroma- and cytokine-independent survival and proliferation. BCR-ABL also induced the expression of myeloid lineage markers, a process that was inhibited by imatinib. By contrast, imatinib did not abolish the stabilization of β-catenin in BCR-ABL transduced mHPC. Transplantation of BCR-ABL-transduced mHPC, but not GFP-transduced mHPC, induced in mice a CML-like disease presented with increased white blood cell counts and splenomegaly. The bone marrow cells from these mice reproducibly transferred CML phenotypes to secondary recipient mice. The diseased bone marrow contains an expanded population of GFP-positive GMP. The effect of imatinib on the leukemogenic potential of these BCR-ABL-positive GMP in vivo is under investigation.

[CONCLUSIONS] Expression of BCR-ABL in the context of mHPC induces leukemogenic traits, including the stabilization of β-catenin, without requirement for additional genetic events. The BCR-ABL-transduced mHPC generates CML-like disease in mice accompanied by the expansion of a BCR-ABL-positive GMP population, similar to that observed in blast crisis CML patients. The stabilization of β-catenin by BCR-ABL is not inhibited by imatinib; consistent with the notion that imatinib cannot eradicate CML stem cells. This murine model can be used to identify therapeutics that targets the CML stem cells.
High-Dose (HD) Imatinib Provides Better Responses in Patients with Untreated Early Chronic Phase (CP) CML.
Authors: Aoki tsuko , Kantarjian Hagop , O’Brien Susan , Talpaz Moshe , Giles Francis , Verstovsek Srdan , Wierda William , Salavado August , Letvak Lauri , Garcia-Manero Guillermo , Jones Dan , Luthra Rajyalakshmi , Shan Jianqin , Cortes Jorge
Published: October 2019

Imatinib (standard dose, SD, 400mg/day) is highly effective in patients with CML in early chronic phase (CP). However, in a phase I study there was clear dose-response correlation and no maximum tolerated dose was identified up to 1000mg/day. Early reports suggested higher response rates with high dose (HD) imatinib (800 mg/day) in this setting. Here we report the long-term follow-up results of high-dose imatinib for patients with CML in early CP. Patients were included from 3 sequential trials: the first included 50 patients all treated with 400mg daily, and 2 subsequent studies included 208 patients all treated at 800 mg daily as the starting dose. There were no differences in pre-treatment characteristics between HD group and SD groups. The median age was 48 years in both groups. Median follow-up was 58 months for SD and 34 months for HD group. Patients treated with HD had a higher rate of complete cytogenetic responses (91% vs 76% in SD group, p=0.002) and these occurred earlier, with 88% achieving a complete cytogenetic response (CGCR) after 6 months of therapy (vs 56% with SD; p<0.00001). The cumulative incidence of major molecular response (MMR) was significantly higher in HD group at 6, 12, 36 and 48 month of follow-up: in HD group, 33%, 58%, 82% and 88% of patients had achieved this response at each time point (vs 2%, 22%, 64% and 67% in the SD group; p<0.00001, <0.00001, 0.01 and 0.002 at respective time point). The cumulative incidence of complete molecular response (CMR) was also significantly higher in HD group at 6, 12, 36 and 48 month of follow-up: in HD group, 8%, 20%, 53% and 66% of patients had achieved this response at each time point (vs 0%, 4%, 27%, and 34% in the SD group; p=0.05, 0.007, 0.003 and 0.0006 at respective time point). Progression-free and transformation-free survivals were significantly better in HD group (p=0.02 and 0.005). Overall survival was excellent in both groups with no difference between them. Grade 3 or 4 hematologic toxicity was more frequent in HD group whereas extramedullary toxicity was similar in both groups. The frequency of ≥ grade 3 anemia, neutropenia and thrombocytopenia in HD group was 12, 35, 29%, respectively while those in SD group were 4, 20, and 12%, respectively. The median actual dose in HD group was 800 mg at 24 months, with only 20% (15/75) of patients taking 400 mg or less at 36 months. We conclude that high-dose imatinib therapy in early CP-CML is associated with higher rates of CGCR, MMR and CMR, and these occur earlier, translating into better progression-free and transformation-free survival. Although HD imatinib is overall well tolerated, there is a higher rate of myelosuppression, most frequently occurring during the first weeks of therapy.
Clinical Characteristics and Outcome of Patients (pts) with T315I Mutation after Imatinib Failure.
Authors: Jabbour Elias , Kantarjian Hagop , Jones Dan , Giles Francis , Borthakur Gautam , O’Brien Susan , Cortes Jorge
Published: October 2019

Point mutations of the Bcr-Abl kinase domain are the most frequently identified mechanism of resistance in patients (pts) with CML who failed imatinib. T315I is an imatinib pocket binding mutation within the Bcr-Abl kinase domain that is highly resistant to imatinib as well as to the novel tyrosine kinase inhibitors (NTKIs) (eg, dasatinib, nilotinib) both in vitro and in vivo. It has thus been suggested that these pts have a very poor prognosis. To define the clinical characteristics and outcome of these pts, we reviewed all pts with this mutations identified at our institution after failure to imatinib. T315I was detected in 15 of 112 patients (13%) harboring kinase domain mutations. Median age was 49 years (range, 34-66 years). At the time imatinib was started, 12 pts were in chronic (CP) and 3 in accelerated (AP). Ten pts had received prior interferon therapy. The best response to imatinib was major molecular response in 1, major cytogenetic response in 5 (complete in 2), and complete hematologic response in 8. One patient was intolerant to imatinib. The median duration of response was 37 months (range, 10–60 months). T315I was identified after a median of 48 months (range, 10–83 months). At the time the mutation was identified, 9 patients had transformed (7 to AP, 2 to BP). Clonal evolution was observed in only 1 pt. Fourteen patients received subsequent NTKIs; 3 of them received both nilotinib and dasatinib. Among them, 1 patient achieved a partial hematologic response with nilotinib that lasted for 3 months and 1 other had T315I identified while in complete cytogenetic response induced by nilotinib that has been sustained for 16+ months. After a median follow-up of 27 months (range, 3–42 months), 11 pts (73%) are alive: 8 are alive with active disease, and 3 patients are alive with ongoing response: 1 in complete molecular remission following allogeneic stem cell transplantation, 1 in partial cytogenetic response following treatment with aurora kinase inhibtor MK0457, and one in sustained complete cytogenetic response with ongoing treatment with nilotinib. Four patients have died of disease progression. Except for previous treatment with interferon (more frequent in patients harboring the T315I mutation; p=0.024) and the lack of response to the NTKI (p=0.001), there was no difference in patients characteristics and previous response to imatinib compared to patients with other mutations.

Similarly, there was no difference in overall survival among patients with T315I mutations or other mutations (p=0.71). In conclusion T315I is a highly resistant mutation to conventional tyrosine kinase inhibitors; however occasional responses can be observed and overall survival may not be as poor as previously reported.
In Vitro and In Vivo Evidence That TSC-22 Is a Putative Tumor Suppressor Gene in Primary Murine and Human Leukemia.
Authors: Yu Jianhua , Yu Li , Hackanson Bjoern , Wei Min , Boyd Zachary , Yokohama Akihiko , Liu Chunhui , Trotta Rossana , Mao Hsiaoyin C. , Becknell Brian , Jaung Michael S. , Liu Shujun , Byrd John C. , Marcucci Guido , Plass Christoph , Caligiuri Michael A.
Published: October 2019

Transforming growth factor-β-stimulated clone-22 (TSC-22) is a gene that has been shown to be silenced in brain and prostate cancer, but its function and the mechanism responsible for this silencing are unknown. We used our model of spontaneous T-natural killer (NK) acute lymphoblastic leukemia (ALL) and discovered that the TSC-22 promoter was methylated resulting in absent expression in seven of eight cases of primary NK-T ALL, but not in cells from normal mice or mice with polyclonal expansion of T and NK cells. We found that TSC-22 was undetectable or minimally expressed in mouse lymphoma cell lines YAC-1 and EL-4 and human leukemia cell lines Jurkat and RPMI 8866, but treatment with the demethylation agent 5-aza-2′-deoxycytidine restored or increased TSC-22 expression. We mapped the TSC-22 promoter and discovered a CPG island in the proximal region and determined that its methylation was responsible for the decreased gene expression. Over-expression of TSC-22 slowed in vitro cell growth and resulted in a dramatic decrease of tumor size in vivo. Finally, TSC-22 expression was found to be absent or substantially reduced in human chronic lymphocytic leukemia and acute myeloid leukemia compared to normal human tissue. Collectively, our data indicate that TSC-22 is silenced via DNA methylation within its proximal promoter, and this silencing appears to contribute to its function as a putative tumor suppressor gene in leukemia. Silencing of TSC-22 can be reversed by 5-aza-2′-deoxycytidine, recently approved by the FDA for the treatment of myelodysplastic syndrome.
Distinct Expression Patterns of Human microRNAs in Myeloid Differentiation and Acute Myeloid Leukemia.
Authors: Isken Fabienne , Steffen Björn , Markus Birgit , Hömme Claudia , Kosel David , Illmer Thomas , Merk Sylvia , Dugas Martin , Berdel Wolfgang E. , Serve Hubert , Müller-Tidow Carsten
Published: October 2019

MicroRNAs (miRNAs) play an important role in differentiation, regulation of cell growth, cell death and potentially cancer pathogenesis. In the current study, we profiled expression of 154 human miRNAs by using a stem-loop reverse transcription and real-time PCR approach in 50 patients with AML and 12 control samples (normal bone marrow (NBM) samples and CD34+ cells). All of these samples were simultaneously analysed by whole genome mRNA microarrays. Out of all miRNAs tested, more than 30 were analyzed in a second, independent group of 48 AML samples and 4 controls. Five miRNAs showed differentiation associated expression changes which were confirmed by in-vitro differentiation of HL-60 cells with all-trans retinoic acid (ATRA) or 12-O-tetradecanoylphorbol-13-acetate (TPA). Overall, we found six miRNAs, including the miR-221 and 222 cluster, that showed substantially different expression between AML and CD34+ cells/ NBM. In class prediction analyses using support vector machines, 92% of the AML samples and 100% of the control samples were correctly predicted. We also looked for differentially regulated miRNAs in FAB subtypes and identified several miRNAs with significantly different expression. For example, high expression levels of miR-26a, 23a and 27b occurred in FAB M5. mRNA levels were analyzed with regard to differences in miRNA expression. Significant negative correlations with specific mRNAs were observed for several miRNAs, for example miR-23b. When mRNA expression patterns were analyzed for dichotomized miRNA levels, close associations between specific miRNAs and gene expression clusters were observed. For example, miR-10a levels were closely associated with the expression of hox genes. Taken together, the distinct expression patterns of miRNAs observed in AML strongly suggest involvement of the described miRNAs in both hematopoietic differentiation and pathogenesis of AML.
G125A Single Nucleotide Polymorphism (SNP) in 5′-UTR of the Tumor Suppressor Gene Bax Affects Its Transcriptional Regulation.
Authors: Saxena Anurag , Bonham Keith , Neuls Evan , Moshynska Oksana
Published: October 2019

Lower expression of Bax protein in various human malignancies is associated with poor response to treatment and shorter disease-free survival. We (Cancer Lett2002; 187:199–205) and others (J Clin Oncol; 23:1514–21) have shown the association of a single nucleotide polymorphism (SNP) in the BAX promoter (G125A) with reduced protein expression and treatment resistance in chronic lymphocytic leukemia (CLL). Using luciferase reporter gene assay we demonstrated that this SNP significantly reduced BAX promoter activity (Oncogene2005; 24:2042–9). Our aim was to determine the effect of this polymorphism on the binding of transcription factors. For electrophoretic mobility shift (EMSA), HeLa and K562 nuclear extracts, and for chromatin immunoprecipitation (ChIP), K562 cells were used to study their ability to bind to a radiolabeled DNA probe corresponding to the BAX promoter region with G nucleotide at the position 125 or bearing G125A SNP. Super-shift assay was performed to determine the transcription factor involved in binding. Competition assays were performed to determine differences in the binding ability of the two probes. A panel of antibodies was tested by super-shift and ChIP assays, non-specific antibodies served as negative controls. Two major band shifts were detected by EMSA. The mobility of the detected complexes was different from those observed with GC1 probe, specific for Sp1/Sp3, suggesting the involvement of transcription factors other than Sp1/ Sp3. This was confirmed in super-shift assay and ChIP assay by incubating DNA probes with Sp1 or/ and Sp3 antibodies. We also found in the competition assays that the cold probes competed differently for binding. The findings provide evidence of the ability of G125A SNP to influence transcription factor binding in vitro (as shown in EMSA experiments) and in vivo (in ChIP experiments).
Leukotrienes Stimulate the Release of Cytokines and Proliferation of Hodgkin Reed-Sternberg Cells.
Authors: Sjoberg Jan , Schain Frida , Tryselius Ylva , Backman Linda , Malec Maria , Porwit-MacDonald Anna , Bjorkholm Magnus , Claesson Hans-Erik
Published: October 2019

Background: Classical Hodgkin Lymphoma (cHL) is characterized by a minority of malignant cells, the so-called Hodgkin- and Reed Sternberg (H-RS) cells, surrounded by inflammatory cells such as eosinophils, macrophages and mast cells. In contrast to other tumors, the malignant H-RS cells constitute only a few percent of the total cells in the affected tissue. Therefore, it is generally believed that various compounds released by H-RS cells and the interaction between H-RS cells and by- stander cells are of great importance in the pathophysiology of cHL disease.

Aim: To characterize the expression and function of cysteinyl leukotriene receptors (CysLTR) in cHL.

Methods and results: We have identified functional CysLT1R in a HL cell line as shown by increased intracellular calcium release upon leukotriene (LT) D4 stimulation (100–500 nM). This response was completely blocked after addition of zafirlukast, a specific CysLT1R antagonist. Immunohistochemical studies of paraffin embedded cHL tissue showed H-RS cells positive for CysLT1R in 12 of 16 cHL tumors. Microarray analysis of laser captured H-RS cells from 3 tumors not only confirmed the expression of CysLT1R transcripts, but also showed expression of the CysLT2R gene (P. Murray, personal communication). The possible role of the CysLT2R in H-RS cells will be discussed. The HL cell line was cultured in the presence of LTD4 (100 nM) to investigate the effects of CysLT signaling in H-RS cells. Real-time RT-PCR analysis showed up-regulation of TNF-α, interleukin (IL)-6, IL-8 and IL-13 mRNA after stimulation with LTD4. Furthermore, the effects of LTD4 on cytokine protein secretion by the HL cells were studied by flow cytometry. The results showed a markedly increased secretion of TNF-α, IL-6 and IL-8 upon LTD4 stimulation. In addition, LTD4 stimulated cell proliferation in a dose-dependent manner.

Conclusion: Since H-RS cells are surrounded by CysLT producing cells (eosinophils, macrophages and mast cells), these results indicate that CysLT signaling could be of importance in the pathogenesis of cHL by contributing to proliferation of the tumor cell population and the disturbed cytokine features of this tumor. This study was supported by the Swedish Cancer Society.
Increased Tumor Infiltrating FOXP3+ Regulatory T-Cells Are Associated with Improved Survival in Classical Hodgkin Lymphoma.
Authors: Kelley Todd W. , Pohlman Brad , Elson Paul , Hsi Eric D.
Published: October 2019

Background: Regulatory T-cells (Tregs), a CD4+, CD25+, FOXP3+ subset, appear to modulate the tumor microenvironment in lymphoma. Using FOXP3 expression as a marker of Tregs, single series have suggested that higher numbers of tumor infiltrating FOXP3+ cells are associated with improved survival in both follicular lymphoma and classical Hodgkin lymphoma (cHL). However, neither of these findings has been confirmed by separate studies and, in fact, contradictory data exists. We sought to evaluate potential correlations between the number of FOXP3+ cells in the tumor microenvironment and patient outcome.

Methods: 98 previously untreated cHL patients, biopsied and treated with curative intent at the Cleveland Clinic, were studied. FOXP3 was assessed by immunohistochemistry (Abcam) and quantitated as the number of positive cells per high power field (hpf), averaged over 5 fields. Histology was evaluated and medical records were reviewed for clinical features (including gender, age, stage, B symptoms, bulk, number of sites and IPS), lymphoma progression and death.

Results: The mean number of FOXP3+ cells (±standard deviation) was 41.7 ± 33.9 cells/hpf, and the median number was 36.4 cells/hpf (range 0–152.0 cells/hpf). Higher numbers of FOXP3+ T-cells were associated with age<45 (P=.02), stage I–II (P=.01), and IPS≤2 (P=.06) [Wilcoxon rank sum test]. 28 of the 98 patients (29%) have progressed and/or died and 20 (20%) have died. The estimated 5-year failure free survival (FFS) and overall survival (OS) are 76%±4% and 86%±4%, respectively. 70 patients remain alive with no evidence of disease at a median follow-up of 8.6 years (range 0.9–19.9 years). Recursive partitioning demonstrated an optimal cutoff of 25 FOXP3+ cells/hpf for analysis of outcome. Overall, 43 patients (44%) had a mean of <25.0 FOXP3+ cells/hpf and 55 (56%) had 25.0 or more. In univariate analysis, higher numbers of FOXP3+ T-cells in the tumor microenvironment were associated with better FFS; estimated 5-year FFS was 91% ± 4% for patients with >25.0 FOXP3+ cells/hpf compared to 81% ± 6% for patients with <25.0 (P=.05, generalized Wilcoxon test). There was also a trend towards improved OS (P=.10). In multivariate analysis, the number of FOXP3+ T-cells remained an independent predictor, even after adjusting for age, stage, and IPS ( P<.05). Kaplan-Meier curves for FFS and OS stratified by FOXP3+ “status” are shown in Figures 1 and 2.

Conclusion: FOXP3+ T cells are a favorable prognostic marker in cHL and reflect the importance of the immune response thus providing rationale for the use of immunomodulatory therapies in these patients.

Failure Free Survival Failure Free Survival

Overall Survival Overall Survival
TARC and MDC Are the Only Chemokines with Highly Increased Levels in Serum of Hodgkin Lymphoma Patients.
Authors: Niens Marijke , Visser Lydia , Jarrett Ruth F. , te Meerman Gerard J. , Poppema Sibrand , van den Berg Anke
Published: October 2019

Chemokines (cytokines with chemoattractant properties) can recruit different subsets of cells and therefore play an important role in the formation and maintenance of the non-neoplastic reactive infiltrate present in Hodgkin lymphoma (HL). The infiltrate consisting of lymphocytes, plasma cells, histiocytes and eosinophilic granulocytes is the most abundant part of the tumor mass in HL and surrounds the minority of neoplastic cells, the so-called Hodgkin-Reed Sternberg (HRS) cells. Several studies have shown that HRS cells and cells in the reactive infiltrate produce multiple chemokines. Especially TARC (CCL17) and MDC (CCL22) are highly produced by HRS cells. Altered serum chemokine levels might be related to HL prognosis or disease activity, since immunological mechanisms are crucial in HL pathogenesis. So far, only TARC and IL-8 levels have been studied in the serum of HL patients. In this study serum levels of nine chemokines including, Eotaxin, Fractalkine, IP10, MCP1, MDC, Mig, MIP1a, RANTES, and TARC were examined in serum of 163 untreated HL patients and 334 healthy controls using ELISA. In a subset of nine patients we also examined serum chemokine levels after treatment. Serum levels of TARC and MDC were significantly increased in 82% and 57% of the HL patient group compared to 12% and 5% in the control group, respectively. Serum Fractalkine and Mig levels did not show a difference between patients and controls, whereas serum levels of Eotaxin, IP10, MCP1, MIP1a, and RANTES were significantly decreased in HL patients. Analysis of the different subtypes revealed that the Nodular Sclerosis (NS) cases contained increased serum TARC and MDC levels compared to the Mixed Cellularity (MC) cases (p-value= 0,000). Serum TARC levels strongly correlated with serum MDC levels (r=0.82, p<0.01). Of the nine patients with serum samples before and after treatment, seven showed decreased serum TARC and MDC levels after treatment. One patient with increased levels before treatment did not show decrease in chemokine levels after treatment and died of disease. The last patient did not have increased chemokine levels before treatment and showed similar low levels in both serum samples. The other chemokines did not show a difference in serum levels in the before and after treatment samples. This is the first study testing a broad set of chemokines in serum of HL patients. Of all chemokines tested, TARC and MDC were the only chemokines with increased serum levels in the vast majority of HL patients and these can be used to monitor treatment efficiency.
The TNF Receptor, CD40 Enters Nucleus through Nuclear Pore Complex and Interacts with c-Rel in Aggressive B Cell Lymphoma.
Authors: Zhou Hai-Jun , Pham Lan V. , Tamayo Archito T. , Lin-Lee Yen-Chiu , Fu Lingchen , Yoshimura Linda C. , Ford Richard J.
Published: October 2019

CD40 is a member of TNF receptor family, which plays important roles in the proliferation, survival and differentiation of lymphocytes. Constitutively active CD40 signaling is one of the hallmarks of aggressive B cell lymphoma development and mediates pivotal functions through its downstream pathways, involving NF-kBs. Recently, our group has reported the nuclear localization of CD40, opening a new paradigm in the functional role of CD40 in non-Hodgkin lymphomas of B cell origin (NHL-B). In this study, we show that CD40 co-localizes and immuno-precipitates with nuclear pore complex (NPC) proteins, which suggests NPC proteins are involved in the classic karyopherin pathway that facilitates the nuclear translocation of CD40 protein. Importantly, our co-immunoprecipitation assays and confocal microscopy have demonstrated that, in NHL-B cells, nuclear localized CD40 interacts with the NF-kB protein-c-Rel, but not p65. Chromotin-IP assay further indicates that CD40 forms complexes with c-Rel on the promoters of several proliferation and survival genes-CD154, Blys/BAFF and Bfl-1/A1 in various NHL-B cell lines. Co-transfection of CD40 and c-Rel in NHL-B cells synergistically enhance Blys promoter-driven luciferase activity while CD40 and p65 do not show such synergy. In normal B cells, transfection of c-Rel has synergistically enhanced the proliferation of B lymphocyte after CD40 ligand stimulation, which results in accumulation of CD40 in the nucleus. Furthermore, co-transfection of c-Rel and wild type CD40 also synergizes the proliferation of NHL-B cells, while co-transfection of c-Rel and nuclear localization signal (NLS) mutated CD40 does not. This study suggests that the nuclear CD40 plays an important role in lymphoma growth and survival mechanisms through its interaction with c-Rel, an oncogene that is amplified in many cases of NHL-B that may contribute to the pathophysiology of the disease process. Modulating the nuclear function of CD40 could provide a new targeted therapeutic approach for lymphoma therapy.
Pre-Clinical Development of Suberoylanilide Hydroxamic Acid (SAHA, Vorinostat ™) for the Treatment of Pediatric Acute Leukemias.
Authors: DeRyckere Deborah , Garrington Timothy P. , Gore Lia
Published: October 2019

Acute leukemia is the most common malignancy of childhood and despite a good outcome for the majority of patients, a proportion of patients has high-risk features, and a very poor prognosis for ultimate cure. As such, novel approaches to therapy and new agents are clearly needed to improve the efficacy and quality of treatment available. We are investigating the utility of several histone deacetylase inhibitors (HDACIs) in the treatment of pediatric acute leukemias. Two HDACIs, suberoylanilide hydroxamic acid (SAHA, Vorinostat™) and MS-275, exhibited broad spectrum anti-tumor activity against a panel of 12 pediatric ALL and 8 AML cell lines representing different cell lineages and stages of development, with clinically achievable IC50 values of approximately 200–900nM. Both agents induced apoptosis in a dose-dependent manner. However, SAHA and MS-275 had differential effects on cell cycle progression and differentiation. Treatment with MS-275 resulted in sustained accumulation of cells in G1 phase and cell line-specific changes in the expression of multiple hematopoietic differentiation markers, including HLA-DR, CD11b, CD13, CD33, and CD61. Differentiation of both AML and ALL cell lines was induced. In contrast, treatment with SAHA either had no effect on cell cycle distribution or caused a transient arrest in G1 phase, which was reversible within 24–48 hours. In addition, only minimal changes in the expression of defined hematopoietic differentiation markers were observed following treatment with SAHA. These data suggest that the anti-leukemia effects mediated by SAHA and MS-275 occur by different biochemical and cellular mechanisms. Consistent with this hypothesis, SAHA and MS-275 exhibited synergistic anti-leukemic activity in vitro in a sequence dependent-manner, with a mean combination index of 0.666 +/− 0.048 based on median effect analysis. Sequence-dependent synergy was noted in a variety of T-cell and pre-B cell lines, including cell lines possessing MLL translocations. Both SAHA and MS-275 exhibited synergistic interactions with drugs representing the topoisomerase II, DNA methyltransferase, and proteosome inhibitor classes based on median effect analysis and/or the universal response surface approach. Detailed data demonstrating the mechanism(s) of HDACI-mediated anti-leukemic synergy and sequence-dependence with these other agents will be presented.
Correlation between WT1 Levels and Chimeric Transcripts in Core Binding Factor AML: Discrepancies Are Not Uncommon AML1-ETO Leukemias.
Authors: Lasa Adriana , Carricondo Maite , Cilloni Daniela , Brunet Salut , Aventin Anna M. , Sierra Jorge , Nomdedeu Josep F.
Published: October 2019

The Wilms tumor gene (WT1) was identified as a tumor suppressor gene coding for a zinc-finger transcription factor. It has been demonstrated that WT1 is overexpressed in acute leukemias and detection of increased levels of WT1 transcripts mainly in peripheral blood, has been associated with clonal growth and relapses. Most AML patients do not have a suitable molecular marker for minimal residual disease (MRD) monitoring. WT1 quantification seems to be an attractive strategy of universal leukemia follow-up. Bone marrow samples from 46 core binding factor (CBF) AML patients were tested for WT1 expression in parallel to AMLI/ETO and CBFb/MYH11quantification. Total RNA was purified using the Trizol reagent (Invitrogen). Chimeric detection was performed following the BIOMED recommendations. The WT-1 mRNA expression was measured by real-time quantitative polymerase chain reaction (RQ-PCR) in an ABI7700 genetic analyzer (Applied Biosystems, Foster City). For WT1 copy number titration the IPSOGEN plasmid was employed (Ipsogen, Marseilles). One hundred and fifty bone marrow (BM) samples from AML1-ETO patients (23 samples at diagnosis and 127 during follow-up) and 195 from CBFbeta-MYH11 cases (21 samples at diagnosis and 174 during follow-up) were included in the study. Bone marrow samples from 6 healthy donors were used to establish the highest acceptable value of WT1 copy number (80 copies). The WT1 expression was significantly increased (up to 3 orders) in bone marrow samples of AML patients at diagnosis compared to BM samples of healthy donors (P < 0.0001). Relapses were observed exclusively in the CBFbeta-MYH11 group and were always preceded by rising amounts of WT1 levels. A good concordance between WT1 levels (80 copies) and prognostically relevant chimeric trancript copy number (1 copy) was detected in 96.23% of the samples. Discrepancies between WT1 and specific fusion genes were observed in 11 AML1-ETO samples and in two CBFbeta-MYH11 cases. In ten follow up AML1-ETO samples with copy values of 1 or less a WT1 result exceeding the 80 threshold was detected. This discordant result was found in a single CBFbeta-MYH11 sample. Our findings suggest that WT1 is a reliable MRD marker in CBFbeta-MYH11 AML. It remains to be investigated the meaning of high titers of WT1 in AML1-ETO acute leukemias.
SNS-595 a Novel S Phase Active Cytotoxic Acts Synergistically with Cytarabine To Reduce Bone Marrow Cellularity and Circulating Neutrophils.
Authors: Arbitrario Jennifer , Kumer Jeffrey , Howlett Anthony , Silverman Jeffrey
Published: October 2019

SNS-595 is a novel naphthyridine analog, a class of compounds not previously used for cancer treatment. SNS-595 is currently in an escalating-dose phase 1 trial in acute leukemia and several phase 2 studies in solid tumors. SNS-595 has a unique mechanism of action; causing double-strand breaks during S phase that are solely repaired through a DNA-PK-dependent pathway. This damage induces DNA damage-repair signals through both p53 and p73 pathways during DNA synthesis and is accompanied by a rapid onset of apoptosis and an irreversible G2 arrest. This mechanism suggests that SNS-595 could interact in combination favorably with other DNA damaging agents that utilize different mechanisms of damage signaling and repair. SNS-595 demonstrates potent anti-proliferative effects on human leukemic cell lines in vitro and is active in LM-3 Jck and CCRF-CEM hematologic xenograft models. Previously, we have shown that administration of SNS-595 results in a dose dependent loss in bone marrow cellularity and reduction in circulating neutrophils in mice (Proc. Amer. Assoc. Cancer. Res.47: 4726, 2006). These data suggest that SNS-595 activity in bone marrow and circulating blood may provide a therapeutic rationale for the treatment of acute leukemias and advanced chronic myelogenous leukemia. In this study we evaluated the effects of SNS-595 on peripheral white blood cells and bone marrow cellularity as a single agent and in combination with cytarabine (Ara-C) and daunorubicin (DNR). SNS-595 dosed once a day at 10 mg/kg IV as a single agent and Ara-C dosed three times a day as a single agent at 20 mg/kg SC, on day 0 and 4, resulted in 44% and 12% reductions in cellularity, respectively. In contrast, combination dosing of SNS-595 once a day at 10 mg/kg IV with Ara-C dosed three times a day at 20 mg/kg SC resulted in a 93% reduction in cellularity. Normal cellularity recovered on Day 12 (8 days post last dose). Furthermore, the SNS-595/Ara-C combination reduced cellularity more than Ara-C dosed at the MTD of 60 mg/kg three times a day SC (42% reduction in cellularity). Circulating leukocyte levels were also reduced including neutrophils that dropped from 1359 cells/ml to 587 cells/ml for SNS-595 dosed at 10 mg/kg IV and 1212 cells/ml for Ara-C dosed three times a day at 20 mg/kg SC on day 8. The co-administration of these two doses resulted in a near elimination of circulating neutrophils (29 cells/ml). Leukocyte counts subsequently returned to normal levels by Day 18 (14 days post last dose). In conclusion, co-administration of SNS-595 and Ara-C reversibly ablates murine bone marrow cells and reversibly depletes circulating neutrophils. These data indicate that SNS-595 has the potential to combine effectively with Ara-C for the treatment of acute leukemias and suggests that additional studies in patients with advanced hematologic malignancies are warranted.
Loss of Heterozygosity (LOH) of the NUP98 Gene Is an Adverse Prognostic Factor in Acute Myeloid Leukemia (AML).
Authors: Plasilova Magdalena , Ommen Hans B. , Jelinek Jaroslav , Issa Jean-Pierre , Riedel Elyn R. , Mathew Susan , Chadburn Amy , Zhou Pengbo , Hokland Peter , Moore Malcolm A.S.
Published: October 2019

The NUP98 gene on chromosome 11p15 encodes a 98kDa protein component of the nuclear pore complex that is involved in the nucleocytoplasmic transport of proteins and RNAs. Chromosomal translocations creating NUP98 fusion genes have been described in various hematologic malignancies including AML and myelodysplastic syndromes (MDS). The molecular mechanisms involving NUP98 gene locus and its function in leukemogenesis remain unknown, however, frequent allelic loss at the 11p locus has been reported in solid tumors and AML. We have evaluated NUP98 gene disruption by LOH in AML subtypes and correlated LOH with clinical outcomes including disease relapse and survival rates. A total of 101 patients with AML and 49 with MDS treated between 2002–2006 were evaluated for LOH at five markers in and around the NUP98 gene. We found a high frequency of 11p15 LOH of the NUP98 region in patients with overt AML (32/101) and a lower frequency in preleukemic MDS patients (4/49). The median follow-up for survivors was 26 months (range 15–43). Patients with NUP98 LOH had a significantly decreased overall survival as compared to patients without NUP98 LOH (median survival 4 and 13 months, respectively, p=0.007). In the subset of patients with no evidence of disease after treatment (n=57), the presence of NUP98 LOH was significantly associated with a decreased disease free interval (5 months vs. 24 months, p <0.0001). The NUP98 disruption was not associated with age, gender, cytogenetic findings, FAB subtype, FLT3-ITD status, prior history of MDS, WBC or platelet counts as confirmed by multivariate statistical model. We did not observe the silencing of the NUP98 gene by methylation of its promoter compared to the p57 gene, located on the same chromosome. In addition to LOH, we screened for chromosomal translocations involving the NUP98 region. In one AML patient we found both NUP98-HOXA9 translocation and LOH. By using the NUP98 antibody that detects both wild type and fusion protein, we found a significantly impaired localization of NUP98 protein and the presence of NUP98 co-localization aggregates within both the nucleus and cytoplasm of one patient, which indicated both NUP98-HOXA9 translocation and LOH. In summary, NUP98 LOH is associated with a worse clinical outcome for AML patients. The presence of NUP98 LOH supports the hypothesis that NUP98 allelic loss may be an important event in de novo leukemogenesis, when leukemia develops after MDS. NUP98 translocations associated with NUP98 fusions contribute to leukemogenesis, particularly in association with NUP98 LOH.
Novel Leukemia Associated Immunophenotype (LAIP) Absent from Normal and Regenerating Bone Marrow Identified by Multiparameter 6-Color Flow Cytometry.
Authors: Guyotat Denis , Olaru Daniela , Flandrin Pascale , Nadal Nathalie , Campos Lydia
Published: October 2019

Flow cytometry analysis of minimal residual disease (MRD) in acute myeloid leukemia (AML) is based on the detection of aberrant phenotypes responsible for the relapse. Until now, all studies were performed by 3 or 4 color immunostaining, allowing the identification of LAIP in 80% of cases. Moreover, no data is available regarding the existence of such phenotypes in regenerating bone marrow. The new generation of cytometers allows the study of 8 parameters that permit a better distinction of malignant from normal phenotypes. In our study we analyzed 20 bone marrow samples from allogeneic donors, 20 ALL regenerating bone marrows after chemotherapy and 53 AML samples at diagnosis. Multiparameter 4 colour and 6 colour flow cytometry was used in order to define antigen combinations which are totally absent or present at very minimal levels in normal and regenerating hematopoiesis. “Blast cells” were gated according to CD45/SSC properties.For the first time we describe by 6 color flow cytometry 47 phenotypes totally absent from “blasts” gate in all normal bone marrow (ex: CD34+DR−117+33−15+, CD34+38+33−56+19−, CD14−DR+4+11B+64+). Another 41 phenotypes were identified as presents at a frequency < 0,05% of total cells (ex: CD34+DR+117−33+15+, CD14−DR+4+11B+64−, CD34+65−56+4−16−). There was no significant difference between normal and regenerating marrows. The 4 color panel of moAbs allowed us to identify only 30 phenotypes presents at a frequency < 0,05% of total cells (ex: CD34+33−13+, CD34+117+11b+, CD34+DR−13+). 53 AML at diagnosis were studied using 6 color immunophenotyping and 58 % of phenotypes described as aberrant or infrequent in normal myeloid hematopoiesis were found in at least one AML at diagnosis in more than 1% of total cells. All AML cases show at least one LAIP but frequently we observed more than one LAIP blast subpopulation in the same sample. Some examples of LAIP observed are CD34+ 38+ 33+ 56+ 19−, CD34+ 38+ 33+ 56− 19+, CD34− DR− 117+ 33+ 15−. In conclusion our results shows that (1) the ability to clearly distinguish leukemic from the healthy cells is considerably increased by 6 color approach (8 parameters analyzed) than 4 color. (2) Furthermore that these aberrant or infrequent phenotypes in normal or regenerating bone marrow samples are identified in AML cases and can be utilized in AML minimal residual disease study. (3) Knowledge of the expression of different markers in normal hematopoietic development provides a frame of reference for identification of abnormal differentiation patterns.