bluuebunny/crossref_metadata_2025 huggingface dataset:
A Novel Cellular Pathway of SCL T-Leukemogenesis Revealed by a Conditional Transgenic Mouse Model.
Authors: Gothert Joachim R. , Brake Rachael , Begley C. Glenn , Izon David J.
Published: October 2019

The acquired activation of stem cell leukemia (SCL) during T-lymphopoiesis is a common event in T-cell acute lymphoblastic leukemia. Here, we generated a novel tamoxifen-inducible transgenic mouse model (lck-ERT2-SCL) to study the cellular targets of acquired SCL activation during T-cell development. Upon tamoxifen treatment we observed the thymic emergence of abnormal, non-cycling CD8+TCRβlow and immature CD4+CD8+ (double-positive, DP) cells displaying increased viability. Unexpectedly, fetal thymic organ culture analysis of lck-ERT2-SCL thymi revealed the development of DP cells before the emergence of CD8+TCRβlow cells, which implied the derivation of CD8+TCRβlow cells from DPs rather than immature CD8 single-positive (SP) thymocytes. Interestingly, histone deacetylase (HDAC) inhibition with trichostatin A (TSA) had a divergent effect on SCL perturbed thymopoiesis: TSA increased T-cell receptor surface expression within DP and CD8 SP cells however did not alter the CD8 shifted CD4/CD8-ratio. Furthermore, we studied the expression of NOTCH1 in SCL induced TCRβlow thymocytes. Strikingly, we found that SCL induced NOTCH1-upregulation in DP TCRβlow cells. We therefore conclude that SCL promotes the emergence of abnormal CD8+TCRβlow cells by an only partially HDAC dependent mechanism from DP TCRβlow cells. Moreover, SCL induced DP TCRβlow cells are characterized by upregulated NOTCH1, which in turn might promote the effect of acquired NOTCH1 mutations during T-leukemogenesis.
Rapid Clearance of PEG-Asparaginase in ALL Patients by an Antibody Against Poly (Ethylene Glycol).
Authors: Armstrong Jonathan , Hempel Georg , Koling Susanne , Chan Linda S. , Meiselman Herbert J. , Fisher Timothy C. , Garratty George
Published: October 2019

Background: Rapid clearance of poly(ethylene glycol)-asparaginase (PEG-asparaginase) has been reported for up to one third of patients treated for acute lymphoblastic leukemia, potentially rendering their treatment ineffective [Hempel G, Lanvers-Kaminsky C, Mueller, HJ, Würtwein G, Boos J. Blood. 2004;11:751A]. Poly(ethylene glycol) is an inert, biocompatible synthetic polymer and has long been claimed to be non-immunogenic; several PEG-conjugated drugs are FDA approved and many other PEGylated agents are in development. However, we have previously reported a 25% occurrence of an antibody against PEG (anti-PEG) in healthy blood donors, which suggests that PEG is an immunogenic agent. [Garratty G. Trans Med Rev. 2004;18:245–56].

Objective: To determine whether an anti-PEG could account for the rapid clearance of PEG-asparaginase.

Methods: This study re-analyzed stored serum samples from pediatric patients who were enrolled in the Acute Lymphoblastic Leukemia Berlin-Frankfurt-Münster 2000 studies. Samples were selected to include 15 subjects who showed undetectable asparaginase activity after receiving PEG-asparaginase (Oncaspar®) and 13 with normal sustained levels of asparaginase activity after treatment with PEG-asparaginase. Sixteen subjects treated with unmodified asparaginase (Medac®) were also included, 8 with negligible asparaginase activity. Sera were tested for the presence of an anti-PEG using two techniques: 1) Serology, by agglutination of PEG-coated red blood cells; 2) Flow cytometry, by analysis of 10 μm PEG beads (Tentagel®-OH) pre-incubated with sera and stained for bound immunoglobulins with fluorescein-anti-human IgG and R-phycoerythrin-anti-human IgM. Testing for anti-PEG was performed in a blinded manner.

Results: For the PEG-asparaginase treated patients, of the 15 who showed undetectable asparaginase activity, 9 tested positive for anti-PEG by serology and 13 tested positive for anti-PEG by flow cytometry. All PEG-asparaginase treated patients with normal sustained asparaginase activity tested negative for anti-PEG. Four sera yielded somewhat unusual results: One weak IgM anti-PEG positive sample showed measurable asparaginase activity and three anti-PEG negative patients had low asparaginase activity, two of which were positive for anti-asparaginase. No relationship was observed between the presence of anti-PEG and serum asparaginase activity for the patients treated with unmodified asparaginase (Medac®).

Conclusions: The presence of anti-PEG was very closely associated with rapid clearance of PEG-asparaginase. Screening and monitoring for anti-PEG would allow for identification of a subset of patients for whom a modified dosing strategy or use of a non-PEGylated drug would be appropriate.
Increased Infection-Related Mortality for Children with Down Syndrome (DS) in Contemporary Children’s Oncology Group (COG) Acute Lymphoblastic Leukemia (ALL) Clinical Trials.
Authors: Maloney Kelly W. , Larsen Eric , Mattano Leonard A. , Friedmann Alison , Devidas Meenakshi , Sather Harland , Hunger Stephen , Winick Naomi , Carroll William L.
Published: October 2019

In prior COG ALL trials, children with DS have had increased treatment-related morbidity without major increases in mortality, when compared to children without DS. Recently, COG opened phase III, randomized trials for the treatment of children with high risk (AALL0232) and standard risk (AALL0331) precursor-B ALL. COG AALL0232 uses an augmented BFM backbone regimen with randomized comparisons of 28 days of prednisone (PDN) 60 mg/m2 vs. 14 days of dexamethasone (DXM) 10 mg/m2 during induction (IND), and high dose methotrexate with leucovorin rescue (HD MTX) vs. escalating IV MTX without rescue plus Peg asparaginase using the Capizzi schedule (CMTX) during a two-month interim maintenance (IM) phase of therapy. Children with DS participated in the induction steroid randomization, but were non-randomly assigned to CMTX arm due to known increased sensitivity to HD MTX. During the first 22 months of accrual to AALL0232, there were 4 deaths among 20 patients with DS, 3 of these 4 deaths occurred in pts receiving DXM in IND yielding an IND mortalaity rate of 15%, which is significantly higher than the 4.3% rate in CCG 1961 (the prior high risk trial). COG AALL0331 utilizes a standard 3-drug IND consisting of DXM 6 mg/m2 for 28 days, vincristine and PEG asparaginase. Post-IND therapy includes a 2 × 2 randomization between intensified vs. standard consolidation, and escalating CMTX combined with augmented delayed intensification (DI) vs. standard IM and DI phases. On AALL0331, there were 6 deaths among 26 children with DS during the first 10 months of accrual. The deaths occurred in IND (3), intensified consolidation (1) and standard DI (2). The IND mortality rate for DS children on AALL0331 is 11.5% compared to 1.3% in CCG 1991 and 0% in CCG 1952 which used identical IND therapy. During DI, patients on CCG 1991 received DXM on days 1–7 and 15–21, whereas those on AALL0331 receive DXM on days 1–21; this difference could contribute to an increased risk of infection in the DS population during the DI phases of the latter trial. The majority of deaths have been due to overwhelming infection with cardiovascular collapse during times of neutropenia. Both COG AALL0232 and AALL0331 suspended enrollment to DS patients pending subsequent therapy changes and expanded supportive care guidelines. This experience emphasizes that children with DS carry a significant risk of infection-related mortality and mandates increased vigilance for early detection and treatment of symptoms suggestive of infection.
Increasing FLT3-ITD Length Increases Expression of bcl-xl in AML.
Authors: Seedhouse Claire H. , Pallis Monica , Grundy Martin , Russell Nigel
Published: October 2019

Internal tandem duplications (ITDs) within the FLT3 tyrosine kinase receptor occur in approximately 25% of patients with acute myeloid leukaemia (AML) and are associated with poor prognosis. The ITDs can range from just three nucleotides to hundreds of nucleotides in length and the duplicated sequence is always in-frame. Recent data has suggested that the size of the ITD may be of importance prognostically and increasing ITD size has been shown to be associated with decreasing overall survival and relapse-free survival1. Very little is known about the biological effects of FLT3-ITDs of differing length and it is possible that such findings would help shed light upon the clinical findings. We have therefore studied various biological parameters in 102 primary AML blast cell samples including FLT3 mutation status, FLT3-ITD length, FLT3 and bcl-xl mRNA expression levels and STAT5 phosphorylation (activation).

38 AML samples had FLT3-ITDs and 64 samples were FLT3-wild type (WT); samples were also screened for D835 point mutations and any samples found to have such a mutation were not included in further analyses, therefore WT denotes neither an ITD nor a D835 mutation. FLT3 mRNA levels were determined by real time PCR and normalised to the housekeeper beta 2 microglobulin (b2M). Samples with a FLT3-ITD had a higher level of FLT3 mRNA than WT samples (p=0.02) and the transcript level of FLT3 was also correlated with phosphorylation of the downstream signalling target STAT5 (measured by flow cytometry; n=22; p=0.02), such an association was not seen in the WT samples (n=23; p=0.37). FLT3-ITD mutations have been shown to activate the anti-apoptotic gene bcl-xl via STAT5 signalling, therefore levels of bcl-xl were also assessed in these samples. A strong correlation was found between increasing levels of FLT3 and bcl-xl transcripts in the FLT3-ITD samples (n=30, p=0.002), this was not found in the FLT3-WT samples (n=50, p=0.42). Bcl-xl levels were found to be dependent upon the level of phosphorylated STAT5 (n=20, p=0.03) and the length of the ITD (n=17, p=0.03) in FLT3-ITD samples. However, as of yet no correlation has been found between STAT5 activation and FLT3-ITD length.

These findings suggest that increasing levels of FLT3 transcripts are associated with increasing activation of the STAT5 signalling molecule and its downstream target bcl-xl. Moreover, the length of the FLT3-ITD was also found to correlate with the level of bcl-xl, although whether this is via a STAT5-dependent mechanism remains to be established. Enhanced levels of bcl-xl may mediate chemoresistance and explain the adverse effect of FLT3-ITD length on outcome in AML.

Bcl-xl mRNA levels correlate with FLT3-ITD length Bcl-xl mRNA levels correlate with FLT3-ITD length
Identification of High-Level DNA Amplifications in AML with Complex Karyotype Using Array-CGH.
Authors: Rücker Frank G. , Bullinger Lars , Miller Simone , Kestler Hans A. , Lichter Peter , Dohner Konstanze , Dohner Hartmut
Published: October 2019

Complex karyotype acute myeloid leukemia (AML), commonly defined as the presence of three or more chromosome abnormalities without specific fusion transcripts, is seen in approximately 10–15% of all AML cases. In this subset of cases, genomic losses and gains are much more frequent than balanced translocations, indicating other mechanisms of leukemogenesis. One possible mechanism is the activation of oncogenes through high-level DNA amplifications. To detect high-level DNA amplifications and to identify corresponding candidate genes, we applied comparative genomic hybridization to microarrays (array-CGH) in 100 cases of complex karyotype AML and correlated the findings with gene expression profiling (GEP) data. For array-CGH a custom-made 2.8k-microarray was used consisting of 2799 different BAC- or PAC-vectors with an average resolution of approximately 2 Mb. Hybridization experiments were performed in a dye-swap setting; significant aberrations were defined as mean plus/minus three times the standard deviation of a set of balanced clones for each individual experiment. In selected cases correlation with global gene expression studies was performed to further delineate candidate genes.

We identified 50 high-level DNA amplifications in 20 different genomic regions. Amplifications occurring in at least two cases mapped to (candidate genes in the amplicon) 11q23.3-q24.1 (n=10; ETS, FLI1, APLP2); 11q23.3 (n=8;MLL, DDX6, LARG, SC5DL); 21q22 (n=5; ERG, ETS2); 9p24 (n=4; JAK2); 13q12 (n=4; CDX2, FLT3, PAN3); 8q24 (n=3; C8FW, MYC); 12p13 (n=2; FGF6, CCND2); 20q11 (n=2; ID1, BCL2L1); and 11q13 (n=2; STARD10, GARP, RAD30, DLG2). To better characterize the genomic architecture of the amplicons, we applied array-CGH using an 8.0k-microarray with an average resolution of approximately 1 Mb. Using this approach highly complex amplicon structures with several distinct amplicon peaks were identified for e.g. the amplified regions in 8q24, 11q23, and 13q12. In addition, parallel analysis of GEP in a subset of 43 of 100 cases displayed overexpressed candidate genes in the critical amplified regions; for some of the genes an oncogenic role has been implicated e.g. C8FW and MYC in 8q24, ETS1, FLI1 and APLP2 in 11q24.1, as well as FLT3 and CDX2 in 13q12. In conclusion, using high-resolution genome-wide screening tools such as array-CGH, a large number of high-level DNA amplifications were identified in AML with complex karyotype suggesting a more general role for protooncogene activation in this AML subset. This high-resolution technique allows the detection of complex amplicon structures with several distinct amplicon peaks pinpointing to selective candidate genes. In addition, correlation with GEP studies facilitates the delineation of overexpressed candidate genes within the amplified regions.
Commonly Dysregulated Genes in Murine Acute Promyelocytic Leukemia (APL) Cells.
Authors: Yuan Wenlin , Payton Jacqueline E. , Holt Matthew S. , Link Daniel C. , Watson Mark A. , DiPersio John F. , James Ley Timothy
Published: October 2019

To identify genes that are commonly dysregulated in a murine model of acute promyelocytic leukemia (mice expressing PML-RARa under control of the murine cathepsin G locus), we first defined gene expression patterns during normal murine myeloid development; serial gene expression profiling studies were performed with primary murine hematopoietic progenitors that were induced to undergo myeloid maturation in vitro with G-CSF. This system provided a method for obtaining highly enriched populations of murine myeloid cells at well-defined stages of differentiation: stem and progenitor cells were enriched on Day 0, promyelocytes were the predominant cell in culture on Day 2–3, mid-myeloid cells were most abundant on Day 4–5, and terminally differentiated myeloid cells were enriched on Day 6–7. These stages were validated using well-characterized “marker” genes for each developmental stage. Many genes were reproducibly expressed in restricted developmental “windows”, suggesting a structured hierarchy of expression that is relevant for the induction of developmental fates and/or differentiated cell functions. Some genes relevant for the pathogenesis of acute myelogenous leukemia (AML) also showed dynamic changes during myeloid differentiation.

We compared the normal myeloid developmental transcriptome to that of APL cells derived from the mCG-PML-RARa knock-in mice. While many promyelocyte-specific genes were highly expressed in all APL samples, 116 genes were reproducibly dysregulated in many independent APL samples. These included 4 classes:genes that were highly expressed in murine APL cells but not in normal myeloid cells at any stage of development,genes normally expressed only in early hematopoietic progenitors (d0) but abnormally expressed in APL cells,genes normally expressed only in late myeloid cells (d7) but also expressed in APL cells, andgenes normally expressed in promyelocytes (d2) but not in APL cells.

Many of these dysregulated genes had previously been linked to oncogenic transformation, including Fos, Jun, Egr-1, TNF, and VCAM-1. However, we found that this set of commonly dysregulated genes was expressed normally in preleukemic promyelocytes from the same mouse model, suggesting that dysregulation occurs as a “downstream” event during disease progression. These studies suggest that the genetic events that lead to APL progression may converge on common pathways that are important for leukemia pathogenesis.
Complex Interaction of the MoeA-N and MoeA-C Domains of Gephyrin Is Required for the Immortalization Activity of MLL-Gephyrin.
Authors: Imamura Toshihiko , Luo Roger T. , Thirman Michael J.
Published: October 2019

More than 40 proteins have been identified as partners of MLL in acute leukemia. The partner proteins are categorized based on their subcellular localization into either the nucleus or cytoplasm. Recent studies have proposed that the mechanism of transformation of MLL-cytoplasmic protein fusion products is mediated by the oligomerization ability of partner proteins. Gephyrin, which is involved in synaptic anchoring of glycine receptor and certain GABAA receptor subtypes, is a rare partner of MLL in patients with AML. Using an in vivo cross linking assay, we confirmed the dimerization activity of a domain within the MoeA-N domain of Gephyrin, which is located at amino acids 456–476. However, we found that this domain was dispensable for immortalization in the methylcellulose colony-forming assay, indicating that the presence of a dimerization domain was not sufficient for transformation. We also observed that the Tubulin binding domain, which is located at amino acids 322–376, was also dispensable. To evaluate which domain of Gephyrin is critical for the immortalization activity of MLL-Gephyrin, we performed a colony-forming assay in methylcellulose and found that both the MoeA-N (amino acids 354–528) and the MoeA-C (amino acids 691–769) domains of Gephyrin are essential for immortalization. In addition, we observed that a MLL- Gephyrin (444–476) deletion mutant formed dimers using the in vivo cross linking assay. To explore the possibility that both MoeA-N and MoeA-C domains are associated with dimer formation of MLL- Gephyrin, we performed co-immunoprecipitation experiments with three fragments of Gephyrin, MoeA-N, MoCF (amino acids 532–690) and MoeA-C. Our data showed that both the MoeA-N and MoeA-C domains interacted with the MoCF domain of Gephyrin. Taken together, these findings show that Gephyrin has multiple dimerization domains, and that the interaction that links the MoeA-N, MoeA-C, and MoCF domains is critical for the immortalization activity of the MLL- Gephyrin fusion protein. Our data indicate that complex patterns of dimerization exist among MLL partners and suggest that specific types of dimerization domains might be critical for MLL-associated transforming activity.
Survivin Is Negatively Regulated by Early Growth Response (Egr)-1 Transcription Factor in Acute Myeloid Leukemia.
Authors: Tamm Ingo , Schmelz Karin , Dörken Bernd , Wagner Mandy
Published: October 2019

Survivin, a member of the inhibitor of apoptosis protein family, is involved in both, inhibition of apoptosis and regulation of cell division. It is expressed in embryonic and fetal tissues as well as in the majority of human leukemias, but is undetectable in normal differentiated adult tissue in vivo. The molecular mechanisms involved in the cancer-specific re-expression of survivin are unclear. In this study, we describe a novel mechanism for over-expression of survivin in AML.

Using electrophoretic mobility shift assays (EMSA), we show that the early growth response (Egr)-1 transcription factor binds to the sequence 5′ GAGGGGGCG 3′ within the human survivin promoter after induction by phorbol 12-myristate-13-acetate (PMA) in vitro. Furthermore, chromatin immunoprecipitation (ChIP) analysis confirmed the specific binding of Egr-1 to the proximal survivin promoter in PMA treated entire leukemia cells.

To further analyze the functionality of the Egr-1 site within the survivin promoter in entire cells, transient transfections of p53 wildtype and mutated cell lines with wildtype Egr-1 expression vector were performed. In these overexpression experiments, mRNA and protein levels of survivin but not of control protein were downregulated after exogenous expression of wildtype Egr-1. Using reporter-gene assays, basal survivin promoter activity was decreased significantly by wildtype Egr-1 transfection, whereas mutant Egr-1 did not change activity of the survivin promoter, implying that Egr-1 specifically blocks survivin expression at the transcriptional level. In addition, Egr-1 over-expression sensitized cells to TRAIL-induced apoptosis.

To investigate the expression pattern of Egr-1 in different AML cell lines and control samples from healthy donors, we analyzed the expression of Egr-1 by RT-PCR. Fitting with the above shown data, Egr-1 was expressed at significantly lower levels in AML cell lines expressing high survivin levels than in healthy control samples with low survivin levels.

Taken together, we show that the transcription factor Egr-1 binds to the human survivin promoter in vitro and in entire cells, downregulates survivin expression independently of p53 and sensitizes cells to TRAIL-induced apoptosis. Since Survivin is often overexpressed in AML, downregulating Survivin expression by activating Egr-1 may be an interesting therapeutic option.
Identification of Meis1 as a Target of CREB in Myeloid Leukemogenesis.
Authors: Esparza Samuel D. , Shankar Deepa , Nelson Stanley , Sakamoto Kathleen
Published: October 2019

The cyclic AMP response element binding protein, CREB, is a leucine zipper transcription factor that induces the expression of genes that regulate proliferation and survival. We previously demonstrated that CREB expression was increased in bone marrow cells from patients with acute leukemia. Transgenic mice that overexpress CREB in myeloid cells develop a myeloproliferative/myelodysplastic syndrome after one year. Although CREB has been implicated in the development of leukemia, the mechanisms by which CREB overexpression leads to a malignant phenotype remain elusive. To identify CREB target genes, we performed microarray analysis with RNA from two CREB overexpressing cell lines, K562 and NFS60. Both murine (NFS60) and human (K562) myeloid leukemia cell lines were stably transfected with CREB under the control of the myeloid-specific HMRP-8 promoter. Meis1, an important transcription factor in hematopoiesis, was found to be upregulated 40-fold in CREB overexpressing cells compared to controls. In the development of granulocytes, Meis1 expression decreases as cells differentiate. Similar to CREB, Meis1 is expressed in CD34+ hematopoietic cells but not in CD34- cells. Meis1 expression has been reported to promote self-renewal of stem cells and inhibit G-CSF-dependent differentiation into granulocytes. We hypothesized that CREB overexpression may lead to persistent expression of Meis1, increased self-renewal, and a block in differentiation of primitive hematopoietic progenitor cells, thereby contributing to leukemogenesis. We first examined Meis1 expression in CREB overexpressing K562 and NFS60 cells. Significantly higher levels of Meis1 were detected in Western blot analysis with lysates from CREB overexpressing K562 cells compared to parental cells. In addition, the degree of CREB expression correlated with the degree of Meis1 expression when evaluated at the protein level. Experiments performed using the NFS60 cell line showed similar results. To investigate Meis1 expression in primary myeloid leukemia cells, bone marrow from patients with acute myeloid leukemia (AML) were analyzed for CREB and Meis1 expression. Samples from three of the four patients had elevated CREB expression. Patients whose cells had the highest levels of CREB also had highest expression of Meis1. The one patient with minimal CREB expression showed no detectible levels of Meis1. Evaluation of cell lines and primary cells using quantitative real time PCR is underway to determine potential differences in CREB and Meis1 expression at the mRNA level. Chromatin immunoprecipitation will also be performed to determine whether Meis1 is a direct target of CREB. Our data demonstrate that CREB overexpression correlates with overexpression of Meis1 in both cell lines and primary AML cells. These results suggest that CREB and Meis1 are co-regulated in AML cells and may contribute to the leukemia phenotype.
Dose-Dense Induction (Sequential-HAM) in Primary Acute Myeloid Leukemia - A Pilot Study of the AML-CG.
Authors: Braess Jan , Staib Peter , Ludwig Wolf D. , Wormann Bernhard J. , Reichle Andreas , Berdel Wolfgang , Buchner Thomas , Gruneisen Andreas , Schmid Christoph , Schmid Christian , Kern Wolfgang , Spiekermann Karsten , Fiegl Michael , Feuring-Buske Michaela , Bohlander Stefan , Heinicke Achim , Sauerland Christ , Unterhalt Michael , Hiddemann Wolfgang
Published: October 2019

Background: Dose intensity is considered one of the prime determinants of antileukemic efficacy in induction treatment of acute myeloid leukemia (AML) - as demonstrated by the superior long-term results of double induction versus conventional induction. In an attempt to further pursue this historically successful strategy an ongoing phase II study of the AML-CG pilots the feasibility of the S-HAM regimen (HD-AraC 3g/m2/12h d1,2,8,9; Mitoxantrone 10mg/m2 d3,4,10,11) in first-line treatment of de-novo AML. In this regimen the interval between the two induction cycles is reduced from 17 days (double induction) to a minimum of 3 days (S-HAM) - thereby increasing dose-intensity more than 2-fold in the critical early phase of treatment.

Results: In the past 18 months 99 patients have been recruited into the trial with a median age of 52 years (range 18 – 78). Of 93 patients evaluable for response the following results were achieved: CR 62%, CRp 24%, PL 6%, ED 8% - resulting in an overall response rate (ORR) of 86%. The early death rate (ED) of 8% and the toxicity profile compared favourably with a historical control group within the AML-CG 1999 study (subgroup: de-novo AML, age less than 60 years, HAM-HAM double induction) with an ED rate of 14% (ORR 68%, persistent leukemia (PL) 18%). The high antileukemic efficacy of S-HAM was also demonstrated by the fact that 88% of patients had a bone marrow blast count of < 10% one week after therapy as compared to less than 48% of patients of the HAM-HAM double induction group. If patients had an adequate blast clearance on day 18 pegylated G-CSF was applied every 10 days until neutrophil recovery. The median time to neutrophil recovery was 30 days after start of treatment with S-HAM which was substantially shorter than following either TAD-HAM or HAM-HAM double induction in the AML-CG 1999 trial (both with a median of 45 days). Since the S-HAM regimen has proven feasible at the present dose level a dose escalation was performed with an additional day of HD-AraC and Mitoxantrone in the first cycle of the sequential regimen.

Conclusion: In the future the appropriate dose level of the S-HAM regimen will then constitute the experimental arm for a randomized comparison of a dose-intensified regimen S-HAM - combining a promising antileukemic activity with significantly reduced duration of critical neutropenia - versus standard double induction for patients younger than 60–70 years in the next generation of the AML-CG studies.
Bone Marow (BM) Involvement in Diffuse Large B Cell Lymphoma (DLBCL): Clinical Impact of Discordant Disease.
Authors: Chhanabhai Mukesh , Connors Joseph , Seville Wayne , Matso Dan , Gascoyne Randy
Published: October 2019

Background and Methods: Most published studies have suggested that survival of de novo DLBCL with discordant BM involvement by small B cell lymphoma is indistinguishable from patients having a negative staging BM. The aim of the study was to investigate the incidence and clinical impact of BM involvement by concordant and discordant B cell lymphoma in patients with DLBCL seen in a single institution over a 5 year period (1\1\2000 – 31\12\2004). The cases were identified from pathology records and BCCA Lymphoid Cancer Database.

Results: The group of interest for this study comprised 652 patients with de novo DLBCL with staging marrow available for review. 60 of 652 (9.2%) of patients with DLBCL had concordant large B-cell lymphoma in their bone marrow. 523 (80.2%) were negative, 16 patients showed what we considered to be atypical lymphoid (ALH) infiltrates lacking definitive features of malignancy. In 50 patients (7.7%) the bone marrow showed discordant histology with predominantly small B-cells, some showing paratrabecular localization. In total there were five cases of TCRBCL in the study, three with marrow involvement. Furthermore, all 54 cases of primary mediastinal B-cell lymphoma (PMBCL) had a negative BM. Of the 652 cases with DLBCL with staging bone marrows available, merging the pathology and clinical databases resulted in 599 patients with complete clinical records. Of these, bone marrows were either not done or deemed inadequate in 101 cases. Therefore, there were 488 patients with DLBCL with an interpretable marrow of which 344 had advanced stage disease. Staging marrows in these patients were negative = 264, positive 41, discordant 28, and ALH 11. The overall survival and progression free survival were strongly affected by the IPI score for these 344 cases (P<0.00001) (see figure). Compared to those without BM involvement patients with concordant large B-cell lymphoma in their BM (n=41) had the worst outcome and those with discordant small B-cell lymphoma in the bone marrow (n=28) had an inferior but intermediate outcome (median survivals (months) = not reached;12 and 20, respectively).

Conclusion: Diffuse large B-cell lymphoma is a heterogenous group of lymphomas as demonstrated by gene expression profiling. Our data suggests that discordant low grade B-cell lymphoma in patients who have coincident DLBCL has a poorer prognosis and the presence or absence of BM disease has clinical significance. Though we had very few cases, TCRBCL show a higher incidence of BM involvement in keeping with the reported literature. The absence of marrow disease in PMBCL is consistent with recent data indicating it is a biologically distinct form of DLBCL.

Figure Figure
Primary Mediastinal Large B-Cell Lymphoma (PMBL) Outcome May Be Significantly Improved by the Addition of Rituximab to Dose-Adjusted (DA)-EPOCH and Obviates the Need for Radiation: Results from a Prospective Study of 44 Patients.
Authors: Dunleavy Kieron , Pittaluga Stefania , Janik John , Grant Nicole , Shovlin Margaret , Steinberg Seth , Raffeld Mark , Staudt Louis , Jaffe Elaine S. , Wilson Wyndham H.
Published: October 2019

PMBL is a distinct clinicopathologic entity characterized by young age, female preponderance, localized disease, prominent sclerosis and CD30+. Gene expression profiling reveals a unique molecular signature, distinct from other DLBCL subtypes, with similarity to classical Hodgkin lymphoma (HL) (J Exp Med 198: 851, 2003). HL is typically CD20 negative whereas PMBL has robust CD20 staining. As with HL, the risk of local failure after anthracycline-based therapy in PMBL has led to routine mediastinal radiation. Given the young median age, female predominance and high cure rates, long-term toxicities from secondary malignancies and coronary artery disease can be life threatening. We prospectively evaluated the role of DA-EPOCH± R without routine radiation in 44 patients with untreated PMBL. The first 18 patients received DA-EPOCH alone and the subsequent 26 received DA-EPOCH+R. DA-EPOCH was administered for 6–8 cycles as described (Blood99: 2685, 2002). Most patients had adverse prognostic features with bulky disease, elevated LDH and extranodal sites, which were balanced among the 2 groups.

Patient Characteristics Characteristics All Patients DA-EPOCH DA-EPOCH-R Total Patients 44 18 26 Gender (F/M) 26:18 (1.44) 10:8 (1.25) 19:9 (1.88) Median age, y (range) 34 (12–70) 34 (20–62) 34 (12–70) Median Mass cm (range) 9.8 (3–19.7) 8.4 (5.1–15.7) 10.2 (3–19.7) Bulky mass > 6 cm 34 (83%) 13 (87%) 21 (81%) ECOG PS > 1 4 (9%) 2 (11%) 2 (8%) Stage III or IV 19 (43%) 9 (50%) 10 (38%) LDH > Normal 32 (73%) 14 (78%) 18 (69%) Extranodal sites 25 (57%) 9 (50%) 16 (63%) Pleural effusion 15 (34%) 4 (22%) 11(42%)

IHC profiling was similar in both groups and consistent with gene expression profiling of PMBL. Analysis of 40 cases showed CD20+ 40/40 (100%), CD10+ 2/30 (7%), BCL-6+ 21/26 (81%), MUM-1+ 10/24 (42%) and high MIB-1 with median (range) of 82% (54–98). At a median potential follow-up of 9.5 and 4.2 years, EFS and OS are shown below for DA-EPOCH and DA-EPOCH-R, respectively. Rituximab was associated with a significantly improved EFS (p=.038) and OS (p=0.023) by 2-tailed exact log-rank test with caveats associated with any non-randomized comparison. Three patients on DA-EPOCH-R had positive PET and biopsy after treatment. One received radiation (event), one recieved salvage chemotherapy and radiation (event), and one no further treatment after biopsy. DA-EPOCH-R is highly effective in PMBL with OS of 100% and obviated the need for radiation/surgery in 23/26 (88%) patients. Rituximab may significantly improve EFS and OS with DA-EPOCH-based treatment. Accrual continues.

Figure Figure
Imbalances of the 9q34 Region Downstream of ABL Gene in CML Are Associated with Blast Crisis.
Authors: Nacheva Elisabeth P. , Brazma Diana , Benson Ruth , Howard Julie , Virgil Anna , Grace Colin
Published: October 2019

Chronic myeloid leukaemia (CML) is a pluripotent haematopoietic stem cell disorder defined by expression of the BCR-ABL fusion gene, a constitutively activated tyrosine kinase. The fusion gene commonly results from formation of the Philadelphia chromosome after a t(9;22)(q34;q11) or related variant rearrangement. The disease follows a biphasic course with an initial benign chronic phase (CP) of 3–4 years treatable with agents such as Imatinib, followed by a rapidly fatal transformation resembling an acute leukaemia (blast crisis, BC). The CML blast crisis has been linked to a number of secondary chromosome changes leading to genome imbalances, such as additional Ph, +8, +9, iso17q. Prognostic scores based on clinical criteria (Sokal and Hasford scores) have been replaced with the discovery of other cytogenetic abnormalities occurring simultaneously at the time of translocation, such as deletions flanking the ABL/BCR breakpoint at the der(9)t(9;22) chromosome that lead to a significantly worse prognosis. We undertook array CGH analysis using an oligo based platform (Agilent) on 46 CML patients (25 CP, 19 BC & 8 in both phases) and 12 CML cell lines revealed a range of recurrent genomic imbalances. Two sets of recurrent cryptic imbalances were identified. These represented both gains and high-level amplifications affecting the 9q34.12 region immediately downstream of ABL gene, as well as losses or gains in the distal part of the 9q34-qter segment. These imbalances were found only in patients at the BC stage. Furthermore, in cases with follow-up samples, the 9q34 imbalances were present in the advanced stage of disease thus confirming their secondary nature. Among the genes found to be amplified in CML/BC is NUP-214, a nucleoporin gene, recently implicated in T-cell acute lymphoblastic leukaemia, thus suggesting an important role for cell clone survival. Confirmation of these amplifications was sought using fluorescent in situ hybridisation (FISH). A dual fusion probe targeting the ABL-NUP214 region was applied to a selection of twenty three patients with confirmed CML and features such as deletion of der(9) chromosome or resistance to treatment with Imatinib, shown to be more prone to variation within the CML phenotypeAmplification of the NUP214 region was found in five patients. These findings represent the first reports of NUP214 amplifications in CML, paving the way for future work.
Identification of a Novel Pathway in BCR/ABL Signal Transduction Involving Akt-Independent Activation of PLC-gamma/mTOR/p70-S6K.
Authors: Markova Boyka , Huber Christoph , Fischer Thomas
Published: October 2019

In BCR/ABL positive CML, defining new, additional therapeutic targets in the pathways, activated by BCR/ABL is critical for the development of new treatment strategies, especially for patients resistant or refractory to Imatinib. While studying the involvement of PI3K/Akt/mTOR signaling pathway in the development of such resistance we have uncovered the existence of additional, Akt-independent mechanism of activation of mTOR/p70-S6 Kinase pathway. Short term treatment with Imatinib (1μM, 4 hours) of the BCR/ABL-positive cell lines LAMA84, AR320, KCL22, K562, Ba/F3-BCR/ABL caused downregulation of p70-S6K phosphorylation and of S6 ribosomal protein phosphorylation without decreasing phosphorylation levels of Akt, as detected by Western blotting using the respective phosphorylation-specific antibodies p-p70-S6K (Thr389), p-S6 (Ser240/244) and p-Akt (Ser473). Inhibition of Akt by the specific inhibitor SH-6 (10μM, 4 hours treatment) did not affect the phosphorylation of p70-S6K and S6. These results were consistent in all analyzed cell lines, and led us to consider alternative mechanism for mTOR/p70-S6K pathway activation. One such mechanism, recently described in FGF9 signaling is a Phospholipase-C-gamma (PLCγ)-controlled Calcium signaling pathway (Wing et al., JBC, 19937–47, 2005), involving Ca/Calmodulin (CaM) and Ca/Calmodulin-dependent Kinase (CaM-K). In all BCR/ABL+ cell lines analyzed we could detect strong PLCγ activation (examined by p-PLCγ-Tyr783 antibody), which was effectively suppressed by Imatinib treatment (1μM, 4 hours). Incubation of the cells for 30 minutes with 10μM U73122, a specific PLC inhibitor, in contrast to the inactive analog U73343, significantly blocked p70-S6K and S6 phosphorylation. The actual mechanism of the Akt-independent mTOR/p70-S6K activation by BCR/ABL is not yet entirely understood. In general, PLCγ activation in turn leads to the activation of different PKC isoform and increase in Ca-dependent signaling. By employing inhibitors of Calmodulin (W7), CaM-K (KN-93) and the PKC inhibitor PKC-412 we are currently studying the participation of these molecules in the pathway. The function of PLCγ/mTOR/p70-S6K pathway for the BCR/ABL driven cells is also analyzed in proliferation (MTT) and apoptosis assays where cells are treated with Imatinib alone or in combination with U73122. Knocking down of PLCγ and other involved molecules is another approach which we will utilize to better understand the necessity/sufficiency of PLCγ for full activation of mTOR/p70-S6K pathway in the BCR/ABL+ cells, as well as the (physical) interaction of PLCγ and BCR/ABL.

In summary, we demonstrate the existence of additional, Akt-independent, PLCγ-dependent mode of activation of mTOR/p70-S6K which operates in BCR/ABL-positive cells. This alternative pathway may prove novel therapeutic targets for CML treatment.
Aberrant Regulation of Wnt/Beta-Catenin Pathway Mediators in Chronic Myelogenous Leukemia Stem Cells.
Authors: Abrahamsson Annelie , Geron Ifat , Gotlib Jason , Durocher Jeffrey , Creusot Remi , Kavalerchik Edward , Goff Daniel , Fathman C.G. , Lilleberg Stanley L. , Giles Francis , Weissman Irving , Jamieson Catriona
Published: October 2019

Background

Recent research suggests that self-renewing leukemia stem cells (LSC) with increased beta-catenin expression are involved in chronic myelogenous leukemia (CML) progression. We investigated whether aberrant regulation of beta-catenin destruction complex genes contributed to the enhanced self-renewal potential of CML LSC.

Methods

FACS Aria purified normal and CML hematopoietic stem cells (HSC), granulocyte-macrophage progenitors (GMP) and lineage positive cells were transduced for 48 hours with lentiviral luciferase GFP and transplanted intrahepatically into newborn RAG2−/−gama−/− mice. At 8 to 12 weeks human CD45+ cells were FACS-purified and transplanted into secondary recipients to establish a bioluminescent CML LSC model. RT-PCR for BCR-ABL was used to confirm CML engraftment. Wnt mediator mutation analysis was performed on cDNA via fluorescent denaturing high performance liquid chromatography (DHPLC) technology and SURVEYOR mismatch cleavage analysis both with the WAVE-HS System (Transgenomic, Gaithersberg, MD). Aliquots of PCR product (3-15 ul) from all samples were scanned for mutations by DHPLC and confirmed by Surveyor mismatch cleavage, and identified with bidirectional sequence analysis on an ABI 3100 sequencer using BigDye V3.1 terminator chemistry. Quantitative RT-PCR was also performed on CML progenitors using destruction complex gene specific primers. FACS analysis was performed with the aid of a FACS Aria to analyze changes in Wnt signaling pathway mediators.

Results

Advanced phase CML was typified by expansion of a GMP population with aberrantly activated beta-catenin expression, enhanced in vitro replating capacity as well as serial transplantation potential in a CML LSC bioluminescent imaging model suggesting that the GMP population was enriched for LSC (Figure 1). A targeted Wnt mutation analysis revealed a mutation in a key component of the beta-catenin destruction complex - glycogen synthase kinase 3beta (GSK) in progenitors from three of six blast crisis CML samples analyzed. Decreased GSK expression was confirmed via FACS analysis using a GSK specific antibody in three separate experiments with CML blast crisis progenitors (Figure 2). Some CML blast crisis progenitors also demonstrated a decrease in axin 2 by quantitative RT-PCR.

Conclusions

Altered expression of Wnt signaling pathway regulators, such as GSK3, may result in increased LSC self-renewal capacity and may represent novel therapeutic targets for advanced phase CML.

Figure 1. Bioluminescent Chronic Myeloneous Leukemis stem cell Model Figure 1. Bioluminescent Chronic Myeloneous Leukemis stem cell Model Figure 2. GSK FACS Analysis. Figure 2. GSK FACS Analysis.
Clinical and Functional Evidence That Drug Transporter Expression Dictates the Response to Imatinib in Chronic Myeloid Leukaemia.
Authors: Wang Lihui , Giannoudis Athina , Lane Steven , Williamson Paula R. , Pirmohamed Munir , Clark Richard E.
Published: October 2019

We have previously shown that imatinib is transported into cells by hOCT1. More recently, we and others have presented data that hOCT1 expression may affect the clinical response to imatinib. Here we extend these observations in two main ways: 1) We have assessed the expression of hOCT1, MDR1 (ABCB1), MRP1 and ABCG2 (BCRP) in a cohort of 70 CML patients, of which 36 achieved complete cytogenetic response (CCR), 4 partial response and 30 no cytogenetic response (NCR). Peripheral blood was taken at baseline and after 4 weeks and 3 months of imatinib treatment. hOCT1, MDR1, MRP1 and ABCG2 gene expression levels were assessed by using real-time quantitative RT-PCR on a LightCycler. Patients were allocated to high-expression and low-expression groups using the median expression as the cut-off level. With a median follow-up of 22 months (from the start of imatinib treatment), patients with high-expression of hOCT1 at baseline had better progression free and overall survival (both p=0.005, log rank test) and a higher probability of achieving CCR (p=0.001, log rank test). MDR1, MRP1 and ABCG2 expression levels at the start of treatment had no correlation with either survival or the achievement of CCR. In addition, amongst responding patients there was a trend for patients with higher MDR1 expression to have a higher incidence of later relapse, typically with the emergence of mutations in the BCR-ABL kinase domain. In 3 of 4 patients who have lost their initial CCR, the MDR1 expression levels were greater than 10% at 3 months and all 4 cases developed BCR-ABL kinase mutation. In contrast, only 4 of 28 patients who had sustained their CCR had greater than 10% of MDR1 expression and none of them had BCR-ABL kinase mutation. 2) In order to further explore the pivotal role of hOCT1 in imatinib uptake into CML cells, we have established stably transfected KCL22 CML cells with high hOCT1 expression. In comparison to untransfected and mock transfected controls, high hOCT1 expressing cells show increased uptake of the fluorescent cation 4-(4-(dimethyl-amino)styryl)-N-methylpyridinium iodide (ASP + ), 14C-TEA and 14C-radiolabelled imatinib (p=0.0002). This enhanced uptake was blocked by the hOCT1 inhibitors amantidine (p=0.0003) and verapamil (p=0.0001). Taken together, the data demonstrate that the baseline expression of hOCT1 (but not MRP1 or ABCG2) is important in dictating the clinical response to imatinib, by means of increased cellular imatinib delivery. In addition, patients with high MDR1 expression may have a higher probability of loss of imatinib response and of kinase domain mutations, and may benefit from closer surveillance of BCR-ABL transcripts and mutations.
Pleural Effusion in Patients (pts) with Chronic Myelogenous Leukemia (CML) Treated with Dasatinib after Imatinib Failure.
Authors: Quintas-Cardama Alfonso , Kantarjian Hagop M. , Munden Reginald , Talpaz Moshe , Bruzzi John , O’Brien Susan , Garcia-Manero Guillermo , Nicaise Claude , Cortes Jorge
Published: October 2019

Dasatinib (SPRYCEL®, BMS-354825) is a multi-targeted kinase inhibitor of BCR-ABL with significant activity in CML after imatinib failure. Dasatinib is well tolerated. The most frequent non-hematologic toxicities are gastrointestinal, rash, and fluid retention syndromes. We evaluated the incidence and outcome of pleural effusion among 131 pts with CML treated in phase I (n=50) and II (n=81) studies of dasatinib after imatinib failure or intolerance at our institution. The median age was 59 years (range, 19 to 81). Sixty-one (47%) pts were in chronic (CP), 30 (23%) in accelerated (AP), and 40 (30%) in blast phase (BP). The daily dose of dasatinib was <70 mg in 15 (11%; 8 bid, 7 qd) pts, 100 mg in 20 (15%; 10 bid, 10 qd), 140 mg in 87 (67%; 66 bid, 21 qd), and >140 mg in 9 (7%; 6 bid, 3 qd). Dasatinib was administered for a median of 42 wks (range, 4 to 120 wks). Pleural effusion occurred in 41 (31%) pts; it was grade 3–4 in 17 (13%). The median time to its development was 5 wks (range, 1 to 107). Effusion occurred in 34% of pts in CP, 34% in AP, and 32% in BP. Effusions were categorized according to the volume of lung involvement: grade 1 (<10% of one lung) occurred in 7 (17%; 5% of total) pts, grade 2 (11–25%) in 17 (42%; 13% of total), grade 3 (26–50%) in 13 (32%; 10% of total), grade 4 (51–75%) in 3 (7%; 2% of total), and grade 5 (> 76%) in 1 (2.5%; 0.7% of total) pt. Grade ≥ 3 effusion occurred in 6 pts with CP and in 11 with advanced phase CML (5 AP, 6 BP). Effusion was more frequent among pts receiving daily doses ≥ 140 mg compared to those treated at < 140 mg (34 of 41 [83%] vs 7 of 41 [17%]; p<0.0001). Effusions were bilateral in 78% and exudative in 78% of pts. Dasatinib was interrupted in 33 (80%) and dose-reduced in 29 (71%) pts. Effusion recurred in 10 (24%) of 41 pts, leading to more than one treatment interruption. Effusion was managed with diuretics in 71%, steroids in 27% (steroids plus diuretics in 12%), thoracentesis in 22% of pts and chest tube in 5%. Dasatinib was permanently discontinued due to recurrent effusion in only 3 pts. In conclusion, pleural effusion is a not uncommon, but manageable complication of dasatinib therapy, particularly among pts in advanced phases of CML receiving dasatinib doses ≥140 mg. In most cases, early identification, temporary dasatinib interruption, the use of diuretics and/or pulse steroids, and subsequent dasatinib dose reduction led to rapid resolution.