bluuebunny/crossref_metadata_2025 huggingface dataset:
Clinical BCR-ABL Peptide Vaccination in Chronic Myeloid Leukaemia: Results of the EPIC Study.
Authors: Rojas Jose M. , Knight Katy , Wang Li-Hui , Clark Richard E.
Published: October 2019

Chronic myeloid leukaemia (CML) is characterised by the BCR-ABL oncoprotein. The amino-acid sequences spanning the junctional region are completely leukaemia-specific. In vitro pulsing of antigen presenting cells elicits immune response to CML cells. Vaccination of CML patients with peptides from this junctional region could therefore elicit/augment immune responses to CML cells. In our Evaluation of Peptide Immunisation in CML (EPIC) study, the patient’s entry requirements were as follows:

first chronic phase of CML, expression of e14a2 (b3a2) BCR-ABL transcript, and prior treatment with imatinib daily (at least 400mg) at a stable dose for at least 6 months.

Each patient received intradermally a cocktail of 3 BCR-ABL peptides:

a 9-mer spanning the e14a2 region, the same 9-mer linked to PADRE (a 15-mer non-natural peptide shown to activate CD4+ T cells), and a 13-mer consensus e14a2 junctional peptide linked to PADRE.

Peptides were administered at either 100 (5 patients), 300 (5 patients), 600 (5 patients), or 1000μg (4 patients) with sargramostim on 6 occasions over 2 months. Immune responses to the vaccine were monitored by IFN-γ and IL-5 ELISPOT assays on peripheral blood mononuclear cells. Molecular responses were assessed by quantitative real-time PCR of BCR-ABL mRNA. At entry no patient showed a detectable immune response to PADRE, but all 19 patients had detectable CD4+ T cells responses within 3 months of commencing vaccination. This indicated that the vaccination protocol was capable of stimulating T cell responses in all 19 patients. Immune responses to the 9-mer BCR-ABL junctional peptide used in the vaccine were detected in 11/19 patients, and demonstrated to be CD8+ T cells by cytokine analysis in flow cytometry. BCR-ABL immune responses were also assessed against a longer 18-mer peptide spanning the whole e14a2 junctional region. CD4+ T cells specific for this 18-mer peptide were detected in 14/19 patients. Interestingly, immunophenotyping indicated that these BCR-ABL-specific T cells were of a memory phenotype (CD45RO+). Serial molecular responses were available for at least 12 months on all cases. Of the 6 patients not in major cytogenic response (MCR) at baseline, molecular improvement was only observed in one case. However 12/13 patients in at least MCR at baseline had at least a 1 log fall in BCR–ABL transcripts, though this occurred several months after completing vaccination. Moreover, vaccination improved the fall in BCR–ABL transcripts in patients who had received imatinib for more than 12 months. These data show that peptide immunisation in CML can elicit anti-BCR-ABL peptide responses in CD4+ and CD8+ T cells. It also demonstrates that BCR-ABL peptide vaccination may improve control of CML, especially in patients responding well to imatinib.
The Immunogenicity of BCR/ABL+ Cells Is Dominated by BCR/ABL-Regulated Antigens and Is Impaired by the Inhibition of BCR/ABL Kinase Activity.
Authors: Scheich Florian , Peschel Christian , Bernhard Helga
Published: October 2019

The inhibition of BCR/ABL kinase activity by imatinib mesylate (IM, STI571, Gleevec®) is the standard therapy for patients with Philadelphia chromosome+ (Ph+) chronic myeloid leukemia (CML). However, the long term treatment with IM or other BCR/ABL kinase inhibitors may be limited due to the development of resistant disease and accumulating side effects. Immunotherapeutical approaches directed against Ph+ CML may overcome these problems. So far, the research to develop an immunotherapy against Ph+ leukemia focused on the BCR-ABL fusion protein, giving the promising opportunity to stimulate cytotoxic T lymphocytes against the joining region segment of p210 BCR/ABL. However, only a limited number of peptides spanning the fusion region is endogenously processed and presented in context with HLA class I molecules. In contrast, the constitutively active BCR/ABL kinase leads to the upregulation and activation of multiple proteins, which may subsequently result in the expression of different, probably leukemia-associated antigens on the cell surface of BCR/ABL-positive malignant cells. In this study, we investigated the immunogenicity of antigens upregulated by BCR-ABL kinase activity, and verified the capacity of these antigens to induce an anti-leukemia T cell response in vitro. We performed CD8+ T-cell stimulations with dendritic cells transfected with mRNA coding BCR/ABL wild-type (WT). Following two stimulations, the proliferating T-cell populations were analyzed for cytokine secretion (IFN-γ) in response to target cells expressing either BCR/ABL WT or a kinase deficient (KD, K1172R) variant of BCR/ABL. With this experimental setting it was possible to compare the immunogenic potential of antigens upregulated by BCR/ABL with the immunogenicity of the BCR/ABL protein itself. We were not able to activate T-cell populations directed against either the breakpoint-region of BCR/ABL or the BCR- or ABL part of the fusion protein. In contrast, we show here that the constitutively active kinase domain of BCR/ABL has a key role in enhancing the immunogenicity of BCR/ABL+ cells. A broad, HLA-dependent T-cell immune response specifically directed against BCR/ABL regulated antigens was detected. The inhibition of BCR/ABL kinase activity by IM significantly impaired the immunogenicity of BCR/ABL+ cells. We found the same T-cell reactivity pattern in several healthy donors and a CML patient. This is the first study demonstrating the major contribution of the BCR/ABL kinase domain to the immunogenicity of BCR/ABL+ cells as T-cell responses against these cells are dominated by BCR/ABL regulated antigens, and not by BCR/ABL itself. These results may contribute to the design of clinical vaccination trials for the treatment CML patients with minimal residual disease, e.g. following successful induction therapy with BCR/ABL inhibitors.
Depletion of Circulating Plasmacytoid Dendritic Cells in Chronic Myelogenous Leukemia and Their Restoration after Interferon-alpha or Imatinib Mesylate.
Authors: Rossignol Alexis , Barra Anne , Roy Lydia , Guilhot Francois , Turhan Ali G. , Gombert Jean-Marc
Published: October 2019

It is currently established that the immune system plays a critical role in the control of chronic myelogenous leukemia (CML). Several cell populations including T cells, dendritic cells (DC) and NK cells have been shown to exhibit potential anti-leukemic activities. The possible role of plasmacytoid dendritic cells (pDC) in this anti-leukemic response has not been explored so far. As pDC are the major source of interferon(IFN)-α in vivo, the frequency and the function of circulating pDC were compared in CML patients and in healthy subjects. Three groups of CML patients were studied:

chronic phase patients (CP) (i.e. at diagnosis or unresponsive to treatment); patients in complete/major cytogenetic remission induced by IFN-α and patients in complete/major cytogenetic remission induced by imatinib mesylate (IM).

Flow cytometric analysis of pDC (assessed by the strong co-expression of CD123 and CD303, formerly BDCA2) showed a dramatic depletion of pDC compartment in CP patients (n = 11) as compared to healthy donors (n = 9) (0.99 versus 5.71 pDC/μL of blood, respectively). Moreover, their functional capacity to produce IFN-α after peripheral blood mononuclear cells (PBMC) stimulation with influenza virus was abolished (883 versus 8201 pg/ml of supernatant, respectively). Patients treated with IFN-α exhibited a complete and long-termed restoration of pDC compartment (4.88 pDC/μL of blood) and of IFN-α production (6932 pg/ml), which was sustained even after the treatment was stopped. In contrast, although IM therapy led to the complete restoration of IFN-α production (6662 pg/ml), its effect on circulating pDC number remained solely partial (2.82 pDC/μL of blood). This last observation and the absence of any linear relationship between pDC number and IFN-α production in patients in complete remission suggested that treatments, and more particularly IM, were able to induce “pDC-independent” pathways of in vivo IFN-α production. Consistently with this hypothesis, we found that pDC-depleted PBMC from patients in cytogenetic remission were able to produce IFN-α, whereas those from healthy donors were not. As a conclusion, our results show for the first time that IFN-α and IM treatments reverse the quantitative and functional impairment of pDC compartment in CP CML patients. They also suggest that these treatments are able to induce differentiation mechanisms leading to the generation of new IFN-α producing cell populations.
MLL Modulates Telomere Length in Mammalian Cells.
Authors: Caslini Corrado , Hess Jay L.
Published: October 2019

In mammalian cells, the telomeric complex at the end of chromosomes consists of several thousand copies of the exanucleotide TTAGGG and associated proteins attached to the nuclear matrix. Chromatin modifying enzymes involved in histone H3/lysine 9 and histone H4/lysine 20 trimethylation, and DNA methylation are known to preserve the telomere heterochromatic structure, length and capping function. Loss of these heterochromatic marks leads to telomere lengthening, most likely through the negative regulation of telomerase or alternative lengthening of telomeres (ALT) mechanisms. The MLL protein is a chromatin modifying enzyme with histone H3/lysine 4 methyltransferase activity, which maintains active transcriptional state of target genes in a large multiprotein complex. Analogously, the yeast’s MLL homologous protein Set1 is part of a multiprotein complex required for maintenance of target genes expression. In addition, Set1 deletion mutants show disruption of telomeric silencing along with telomere shortening or lengthening, respectively in budding and fission yeast. This raised the question of whether MLL, like Set1, plays a role in epigenetic maintenance of telomeric heterochromatin. Here, using chromatin immunoprecipitation (ChIP) analysis, we show that MLL associates with the heterochromatic complex at telomeres of primary and transformed human cell lines. ChIP analysis of cell lines conditionally expressing Flag-tagged MLL chimeric proteins and deletion mutants shows the amino terminus of MLL, which confers association to the nuclear matrix, is responsible for targeting to the telomeric complex. MLL associates with the telomeres of telomerase and ALT positive cell lines in amount that is proportional to the telomere length, as revealed by Southern blot terminal restriction fragment analysis. Moreover, immunoprecipitation analysis evidenced the association of MLL with the terminal-repeat binding factor TRF2, a protein known to play a key role in telomere capping, and indirect immunofluorescence analysis showed MLL and TRF2 colocalization at ALT-associated PML nuclear bodies.

In search for possible biological functions of MLL at the telomeric complex, we found abnormally longer telomeres in Mll-null mouse embryonic stem (ES) cells and fibroblasts (MEFs) than in wild-type control cells. In Mll-null MEFs, a significant telomere shortening was obtained by stable reexpression of an MLL allele. In addition, we found that in aging human cells the MLL binding to the telomeric complex is abrogated by the progressive telomere shortening due to telomere attrition, suggesting a possible involvement of MLL in signaling for replicative senescence.
Acetylation of a Conserved Lysine Residue within Activation Domain One of E2A Transcription Factors Plays a Role in Transcriptional Regulation.
Authors: Hyndman Brandy D. , Bayly Richard , LeBrun David P.
Published: October 2019

The E2A locus encodes transcription factors, called E12 and E47, involved in lineage-specific cellular differentiation. The locus is also involved in chromosomal translocations associated with acute lymphoblastic leukemia, the most common of which results in expression of the oncoprotein E2A-PBX1. We showed recently that direct interaction between E2A and the histone acetyl-transferase (AT) and transcriptional co-activator proteins CBP/p300 is required in leukemogenesis by E2A-PBX1. E2A proteins have also been shown to interact with another AT/co-activator, p/CAF. Interaction with these AT proteins results in acetylation of E2A proteins themselves. Here we map the acetylated lysine residues within the oncogenic portion of E2A proteins and begin to elucidate some functional correlates of E2A acetylation. Our results indicate that the isolated AT domain of p/CAF as well as full-length p/CAF were capable of acetylating E2A. Interestingly, full-length p300 was capable of acetylating E2A while an isolated AT domain was unable to acetylate E2A, suggesting that additional domains of CBP/p300 are required to mediate E2A acetylation. We demonstrate that both p300 and p/CAF can interact directly with E2A, independent of the known interaction between p300 and p/CAF. These co-activators do, however, appear to co-operate to achieve maximal E2A acetylation. Mutagenesis-based mapping studies indicate that several lysines are substrates for acetylation. Of particular interest, a conserved lysine residue (K34) located within the N-terminal transcriptional activation domain (AD1) is acetylated in vitro by p/CAF. K34 is located within a GKXXP consensus sequence, suggested to be a recognition motif for acetylation by several AT enzymes including CBP/p300 and p/CAF. Conservative replacement of K34 with arginine (i.e., K43R) substantially impairs transcriptional activation of a luciferase reporter gene by E2A, suggesting that post-translational modification of this residue may play an important functional role. Consistent with a role for acetylation, relative to some other lysine-dependent modification, we were unable to demonstrate sumoylation or ubiquitination of the N-terminus of E2A. Therefore, we have found that acetylation by AT/co-activator proteins contributes to transcriptional regulation by the functionally critical N-terminal activation domain (AD1) of E2A proteins. The mechanisms by which this acetylation event is regulated and how it contributes to target gene induction by E2A are not clear. It seems plausible that the acetylation status of AD1 could be determined by upstream signaling events and acetylation of E2A could modulate interactions with transcriptional co-regulators, DNA or chromatin. Further studies to investigate these possibilities are underway. In particular, results using an amino acid substitution that mimics acetylation of AD1 (K34Q) will be presented.
Decitabine and Vitamin D3 Differentially Increase C-Jun, PU.1, and Sp1 Hematopoietic Transcription Factors To Induce Monocytic Differentiation.
Authors: Koschmieder Steffen , Agrawal Shuchi , Radomska Hanna S. , Huettner Claudia S. , Tenen Daniel G. , Ottmann Oliver G. , Berdel Wolfgang E. , Serve Hubert L. , Mueller-Tidow Carsten
Published: October 2019

Standard chemotherapy is not curative for many patients with acute myeloid leukemia (AML). New treatment strategies combining demethylating agents, such as decitabine, and drugs that induce myelomonocytic differentiation (i.e. Vitamin D3) may reestablish functional hematopoiesis in these patients.

We studied the effects of decitabine alone or in combination with Vitamin D3 (VD3) on promonocytic U937 cells and Ficoll-enriched blast cells from patients with AML and analyzed their impact on cell growth and viability (assessed by trypan blue exclusion), surface marker expression (FACS analysis of CD11b, C14), and the expression and function of target molecules (Northern blot, Western blot, EMSA). Doses ranging from 0.1 to 1 uM of decitabine induced CD11b and CD14 surface expression 3- to 18-fold and 3- to 4-fold, respectively. 3 to 100nM of VD3 induced CD11b and CD14 expression 1.5- to 10-fold and CD14 1.5- to 8-fold, resp. Interestingly, decitabine synergized with VD3 to increase CD11b and CD14 surface marker expression up to 73-fold and 11-fold, resp., and to induce morphologic differentiation of U937 cells along the monocytic lineage. The mechanisms of decitabine- and VD3-induced monocytic differentiation are currently unclear. Therefore, we investigated the effects of the two drugs on several transcription factors that have been implicated in monocytic differentiation. Northern and Western blotting showed that decitabine induced transcription of c-jun but not PU.1, while VD3 increased PU.1, IRF8, and C/EBPbeta but not c-jun. Monocytic target promoters such as the CD11b promoter have been shown to be activated by a complex containing PU.1 and c-jun. Using electromobility shift assays, we now demonstrate increased DNA binding of nuclear proteins from decitabine- and VD3-induced U937 cells to the CD11b promoter. In addition to the effects seen with the CD11b promoter, we investigated whether the myeloid transcription factor Sp1 played a role in decitabine- and VD3-induced CD14 expression. Indeed, we found that mithramycin A, a specific inhibitor of Sp1, inhibited both VD3- and decitabine-induced upregulation of CD14, which is in line with previous data showing that Sp1 is critical for CD14 promoter activity. Induction of CD11b and/or CD14 by decitabine and/or VD3 was confirmed in primary AML patient samples at the time of diagnosis.

In conclusion, decitabine synergizes with Vitamin D3 to induce CD11b and CD14 expression, likely by enhancing PU.1/c-jun and Sp1 transcriptional activity.
Rexinoid-Triggered Differentiation and Tumor-Selective Apoptosis of AML by Protein Kinase A-Mediated De-Subordination.
Authors: Schiavone Ettore Mariano , Altucci Lucia , De Simone Mariacarla , Ferrara Felicetto , Rossin Aurélie , Hirsch Oliver , Nebbioso Angela , Vitoux Dominique , Wilhelm Emmanuelle , Guidez Fabien , Zelent Arthur , Grimwade David , de Thé Hugues , Gronemeyer Hinrich
Published: October 2019

Apart from PML-RARα acute promyelocytic leukemia all other acute myeloid leukemias (AML) are unresponsive to retinoid differentiation therapy. However, in our study we show that elevating the levels of cyclic AMP (cAMP) confers onto retinoid X receptor (RXR)-selective agonists (“rexinoids”) the ability to induce terminal granulocyte differentiation and apoptosis of all-trans retinoic acid-resistant and insensitive AML cell lines and patients’ AML blasts. Protein kinase A activation leads to co-repressor release from the RAR subunit of the RAR-RXR heterodimer, resulting in “de-subordination” of otherwise silent RXR, which acquires transcriptional competence in response to cognate ligands. Rexinoid-cAMP induction of endogenous RARβ is blunted in mouse embryo fibroblasts lacking RARs, but re-introduction of exogenous RARα re-establishes responsiveness, thus confirming that the RARα-RXR heterodimer is the rexinoid mediator. The apoptogenic effect of this treatment involves enhanced expression of the death receptor DR5 and its cognate ligand, the tumor necrosis factor-related apoptosis inducing ligand (TRAIL), both of which are known to induce apoptosis in a tumor cell-selective manner and lead to the activation of initiator caspases. Immunohistochemistry confirmed induction of TRAIL and DR5 in AML patient blasts cultured “ex vivo”. AML patients’ blasts responded to rexinoid-cAMP combination treatment with induction of maturation and apoptosis, independent of karyotype, immunophenotype, and FAB classification. Clonogenic assays revealed complete inhibition of blast clonogenicity in four out of five tested samples. Indeed, it is known that cAMP levels can be elevated also by treating cells with 3′, 5′-cAMP phosphodiesterase inhibitors (PDEi’s). These observation and the clinical availability of the corresponding drugs provide a rationale for initiating clinical studies addressing the efficacy of combinatorial PDEi-rexinoid therapy in AML patients. Together with our recent finding that a very promising class of epigenetic anti-tumor drugs operates through activation of TRAIL expression (Nat Med2005, 11(1):7784), the possibility to target both the ligand (TRAIL) by HDAC inhibitors and the cognate receptors (DR4, DR5) by the above described rexinoid crosstalk may represent a promising therapeutic option which might lead, despite the genetic, morphologic, and clinical variability of AML, to a novel therapeutic option for AML patients by inducing a tumor-selective death pathway.
Rig-G Protein Links Retinoid and Interferon Signal Pathways To Inhibit Cell Cycle through Increased p27 and p21 Protein Levels.
Authors: Tong Jian-Hua , Xiao Shu , Li Dong , Zhu Hai-Qing , Song Man-Gen , Jia Pei-Min , Pan Xiao-Rong , Chen Sai-Juan , Chen Zhu
Published: October 2019

Over the last two decades, the molecular mechanisms of all trans retinoic acid (ATRA)-induced differentiation of acute promyelocytic leukemia (APL) cells have been greatly explored The Rig-G gene was first isolated from an ATRA-treated APL cell line NB4. The expression of Rig-G was undetectable in untreated NB4 cells, but could be dramatically induced by ATRA treatment. Interestingly, database research showed that Rig-G represented a member of interferon-inducible (IFI) gene family. Moreover, a synergistic induction of Rig-G mRNA in NB4 cells by ATRA and interferons (IFNs) indicated a possible role of Rig-G in crosstalk between these two signaling pathways. In this work, we show that Rig-G is indeed a direct target of STAT1 protein, a critical transcription factor in regulating IFN responses, and involved in both ATRA and interferon pathways. By stable transfection of Rig-G in U937 cells, we find that Rig-G protein can significantly accumulate the cells at G1/S transition and inhibit cell proliferation through enhancing some important cell cycle regulators. Further studies demonstrate that Rig-G can physically interact with JAB1 (Jun activating domain binding protein). It can decrease JAB1-enhanced AP-1 transactivities through preventing JAB1 from entering the nucleus, and impair JAB1-dependent p27 nuclear export and degradation, resulting in a nuclear accumulation of p27. In addition, we show that Rig-G can also downregulate c-Myc followed by an upregulation of p21, another major negative player of cell growth. Taken together, it seems that Rig-G may be one of the first experimentally proven molecular mediators responsible for IFNs-dependent growth-suppressive responses, and it can link both IFN- and ATRA-triggered intracellular pathways to synergistically inhibit cell growth in many cell types.
Effect of Germline Polymorphisms on Clinical Outcome in Hodgkin’s Lymphoma (HL).
Authors: Gaya Anna , Urbano-Ispizua Alvaro , Navarro Alfons , Pons Aina , Abrisqueta Pau , Martinez Carmen , Granell Miquel , Martinez Antonio , Lopez-Guillermo Armando , Montserrat Emili , Monzo Mariano
Published: October 2019

In this study, we investigated whether distinct patterns of functional genomic polymorphisms in genes involved in drug metabolic pathways (GSTT1, GSTP1, GSTM1, SULT1C2, TOP2A, SXR-1), DNA repair (XPD, XPA, ERCC1, ERCC5, XRCC1, XRCC4, XRCC5), apoptosis and inflammatory cytokines (FAS, FASL, IL-10), and multidrug resistance (MDR1-2, MDR1-6) predict clinical outcome in patients with HL. One hundred and twenty-seven adult patients (median age, 33 yrs; range, 15–80; males 53%) diagnosed with HL at a single institution between September 1995 and June 2005 have been studied. Seventeen patients (13.4%) were HIV+. Distribution according to histological subtypes was: nodular sclerosis (56.7%), mixed cellularity (20.5%), lymphocytic predominance (5.6%), and lymphoid depletion (5.6%). Epstein-Barr Virus (EBV) was present in 35.4% of the samples. First-line treatment consisted of CMOPP/ABV (39.4%) or ABVD (52.8%). Allelic discrimination of single nucleotide polymorphisms (SNPs) (ABI Prism 7500; TaqMan) of DNA obtained from formalin fixed paraffin embedded lymph nodes was performed. The characteristics considered were: age (<45 years vs. ≥45), ECOG, Hasenclever prognostic index (≤1 vs 2–4 vs ≥5), HIV status, Ann Arbor (I–II vs. III–IV), WHO histological classification, bulky disease, EBV (LMP1+ vs LMP1−), ESR (EORTC criteria), and polymorphisms of the above-mentioned genes. Clinical outcomes analyzed were probability to achieve complete remission (CR), toxicity due to the treatment, relapse rate, disease-free survival (DFS) and overall survival (OS). Out of 127 patients, 101 (79.5%) achieved CR, 7 (5.5%) partial response, 9 (7.1%) were chemoresistant, and 5 (3.9%) died during the initial treatment. After a median follow-up of 43 months (1–128), OS was 81.1% and DFS 62.6%. In the multivariate analysis, the only adverse prognostic factor for the achievement of CR was FASL -844 T>C polymorphism (RR=1.7; p=0.01) and the only adverse prognostic factor for relapse was IL-10 -1082 A>G polymorphism (RR=2.1; p=0.02). A lower probability of DFS was associated with HIV+ status (RR=11.4; p=0.03), and a lower probability of OS to higher Hasenclever prognostic index (RR=2.3; p=0.02). Moreover, a close association between Asp5Glu genotype of the phase II drug-metabolizing enzyme SULT1C2 and pulmonary toxicity was found; thus, all eight patients with pulmonary toxicity due to bleomicine had the wild type SULT1C2 genotype (p=0.006). In conclusion, germline polymorphisms in FASL, IL-10 and SULT1C2, which can easily be analyzed in paraffin embedded samples, have prognostic value in patients with HL.
Absolute Lymphocyte Count Is a Novel Prognostic Indicator in Acute Leukemia: Implications for Risk Stratification and Future Studies.
Authors: De Angulo Guillermo R. , Yuen Carrie , Palla Shana , Anderson Peter M. , Zweidler-McKay Patrick A.
Published: October 2019

Background: Despite improving outcomes, 25–50% of children and young adults with acute leukemia still relapse and most salvage rates are discouraging. Additional prognostic factors, particularly those that represent host factors, may further stratify patients and decrease relapse rates.

Purpose: To determine if absolute lymphocyte counts (ALC) during induction chemotherapy can improve current risk stratification and predict relapse-free survival (RFS) and overall survival (OS) in children and young adults with acute lymphoblastic leukemia (ALL) and acute myeloid leukemia (AML).

Methods: We analyzed 160 consecutive cases of de novo ALL and AML patients 1–21 years of age, treated at the University of Texas M. D. Anderson Cancer Center from 1995–2005. Age at diagnosis, initial WBC, bone marrow blast % on days 0 and 7, were analyzed with ALC on days 0, 15, 21 and 28 of induction therapy.

Results: ALC during induction therapy is a significant independent predictor of RFS and OS in young adults and children with either ALL or AML. Specifically, an ALC <350 cells/mcL on day 15 of induction therapy for ALL significantly predicts poor 6-year OS (52% vs. 87%, p=0.015; HR=4.2, Figure 1A) and RFS (46% vs. 80%, p=0.001; HR=4.8, Figure 1B). Similarly, an ALC of <350 cells/mcL on day 15 of induction therapy for AML predicts poor 6-year OS (35% vs. 86%, p=0.033; HR=4, Figure 1C). ALC-15 remains a significant predictor of OS and RFS after adjusting for age at diagnosis, initial WBC and bone marrow response on day 7 (p=0.013; HR=6.3, and p=0.003; HR=6.3, respectively) in multivariate analysis (Table 1). Importantly, ALC-15 defines a subgroup of half of our AML patients and predicts an excellent 5-year OS of 86% (p=0.033, Figure 1C). Conversely, prolonged lymphopenia predicts that 16% of young AML patients will have a dismal 5-year RFS of 14% (p=0.004, Figure 1D). Finally, ALC-15 <350 cells/mcL is able to predict 70% of relapses in both ALL and AML patients. One possible algorithm could identify half of AML patients with a predicted OS of 86% simply by measuring the ALC-15. Those patients with a low ALC on day 15 would be assessed at day 21 and 28 and those with persistent lymphopenia would be predicted to to have an RFS of 14% and would be stratified to receive intensified and/or experimental therapy.

Conclusion: We demonstrate that ALC can identify patients at high and low risk for relapse early in the course of treatment for ALL or AML. Our data indicates that ALC is both independent of and a more powerful predictor than age at diagnosis, initial WBC and bone marrow response on day 7. This routine measurement could enhance current risk-stratification and lead to improved outcomes in young patients with acute leukemias.

Figure 1 Figure 1. Table 1 Multivariate Analysis of ALC, Age, WBC, Bone marrow response and Survival
The Gene Expression Profile of T-Cell Prolymphocytic Leukemia with inv(14)(q11q32) Reflects the Pattern of Chromosomal Imbalances.
Authors: Dürig Jan , Bug Stefanie , Klein-Hitpass Ludger , Boes Tanja , Jöns Thomas , Dührsen Ulrich , Siebert Reiner
Published: October 2019

T-cell prolymphocytic leukemia (T-PLL) is an aggressive lymphoma derived from mature T-cells, which is in most cases characterized by the presence of an inv(14)(q11q32) and a characteristic pattern of secondary chromosomal aberrations. DNA microarray technology was employed to compare the transcriptomes of eight immunomagnetically purified CD3+ normal donor derived peripheral blood cells with five highly purified inv(14)-positive T-PLL blood samples. In comparison between the two experimental groups 740 genes were identified as differentially expressed including functionally important genes involved in lymphomagenesis, cell cycle regulation, apoptosis and DNA repair. Notably, the differentially expressed genes were found to be significantly enriched in genomic regions affected by recurrent chromosomal imbalances. Up-regulated genes clustered significantly on chromosome arms 6p and 8q and down-regulated genes on 6q, 8p, 10p, 11q and 18p. High-resolution copy-number determination using SNP-chip technology in twelve inv(14)/t(14;14)-positive T-PLL including those analyzed for gene expression refined chromosomal breakpoints as well as regions of imbalances. In conclusion, combined transcriptional and molecular cytogenetic profiling identified novel specific chromosomal loci and genes which are likely to be involved in disease progression and suggests a gene dosage effect as a pathogenic mechanism in T-PLL.
Parallel Molecular and Immunological Monitoring of Minimal Residual Disease in Adult Acute Lymphoblastic Leukemia (ALL) Patients.
Authors: Parovichnikova E.N. , Davidyan J.R. , Galtzeva I.V. , Surin V.L. , Vorobiev I.A. , Gretzov E.A. , Isaev V.G. , Savchenko V.G.
Published: October 2019

Though the initial response to induction therapy, indicating rapid tumor clearance, is currently considered to be one of the most important prognostic factors, detection of submicroscopic levels of minimal residual disease (MRD), by means of PCR or immunophenotyping after induction and during different stages of treatment, has also become important in predicting outcome in ALL patients. We initiated a study using parallel PCR and flow cytometry to monitor MRD in adult acute lymphoblastic leukemia (ALL) patients treated by standard approach. Bone marrow samples were analyzed at diagnosis, after pre-phase (one week of steroid therapy to determine steroid responsiveness), at 8-weeks post induction, at consolidation, before maintenance (+7–8 mo), and during maintenance for the detection of clonal IgH, TCR-gamma rearrangements by PCR (The level of detection sensitivity is 10−3 to 10−4). Specific markers were identified for 88% of the patients (30/34); prolonged monitoring was carried out in 22 patients with follow up from 2 to 24 months. Fifteen of those patients were analyzed by three-color flow cytometry analysis with a panel of 20 monoclonal antibodies for patient-specific aberrant leukemia-associated phenotype (LAP) at diagnosis and before maintenance (10−4). Contrary to PCR analysis, LAPs were detected in all patients. The most common LAPs were CD19/34/10, CD 19/34/TdT, and CD19/34/IgM. Clinical CR was achieved in 85% of patients. Prednisone resistance was detected in 71% of patients. MRD was detected by PCR in all patients after pre-phase, in 21/22 (95%) after 1st induction phase, in 11/15 (73%) after 2nd induction phase, in 10/15 (67%) after consolidation, and in 8/14 (57%) before maintenance. Flow cytometry data coincides with molecular parameters: only 4 of 10 patients examined before maintenance (+7–8 mo) had no cells with LAP. Comparing our parallel measurements we have noted discrepancies in 3 of 10 cases. Two immunologically positive patients were PCR-negative, and one PCR-positive patient was immunologically negative.

All patients (n=3) whose PCR probes were positive more than twice, relapsed at different time points; no relapses were noted in once positive patients (n=3), and 1of 8 PCR-negative patients relapsed. Our data indicate that both methods are effective and relevant in detection of MRD. They show very slow tumor clearance in ALL patients. MRD positivity reflects the higher relapse probability. More patients can be followed for MRD with flow-cytometry, due to higher detection rate of LAP.
A FLT3 Gene-Expression Signature Outperforms FLT3 Status in Predicting Clinical Outcome for Patients with Normal Karyotype AML.
Authors: Bullinger Lars , Dohner Konstanze , Stirner Christoph , Rucker Frank G. , Frohling Stefan , Kim Young H. , Schlenk Richard F. , Tibshirani Robert , Dohner Hartmut , Pollack Jonathan R.
Published: October 2019

In acute myeloid leukemia (AML), cyotgenetics is used to stratify cases for appropriate risk-adapted therapy. The largest subset of patients, those with normal karyotype AML (NK-AML), comprises a heterogeneous group with intermediate prognosis. To improve outcome prediction for this group, we carried out gene-expression profiling of a set of 65 clinically-annotated NK-AML cases. In exploratory studies, an outcome predictor derived from semi-supervised analysis comprised genes mainly correlated with the presence of FLT3 (fms-related tyrosine kinase 3) internal tandem duplication (ITD) activating mutation, itself a prognostic factor. We therefore sought to directly define and characterize a gene-expression predictor of FLT3-ITD mutation status. Using a supervised analysis (Prediction Analysis of Microarrays), we identified an optimal 25-gene FLT3 signature, which in an independent set of 73 NK-AML cases was a significant predictor of clinical outcome, notably outperforming FLT3-ITD mutation status itself. We speculate that the signature identifies cases with alternative genetic or epigenetic changes that phenocopy FLT3-ITD, and the signature genes provide a starting point to dissect these pathways. Our findings underscore the prognostic relevance of FLT3-ITD in NK-AML, and indicate the potential clinical utility of a gene-expression based measure of clinically-relevant FLT3 pathway activation.
Diagnostic and Prognostic Significance of Tryptase in Clinical Hematology: A Single Center Experience from 1070 Patients.
Authors: Sperr Wolfgang R. , El-Samahi Aischa , Girschikofsky Michael , Winkler Stefan , Semper Hans , Agis Hermine , Sillaber Christian , Endler Georg , Rumpold Helmut , Lutz Dieter , Valent Peter
Published: October 2019

Recent data suggest that tryptase is expressed in neoplastic cells in various myeloid neoplasms. In this study, serum tryptase levels were determined by a fluoroenzyme-immunoassay in 165 healthy subjects, in 925 patients (pts) with hematologic malignancies (myeloproliferative disorders, MPD, n=150; myelodysplastic syndromes, MDS, n=233; acute myeloid leukemia, AML, n=317; acute lymphatic leukemia, ALL, n=26, Non Hodgkin′s lymphomas, NHL, n=30; systemic mastocytosis, SM, n=89), and in a large cohort of control cases (pregnant women, n=11; hepathopathy, n=7; renal failure, n=18; helminth infections, n=29; reactive leukocytosis/thrombocytosis or idiopathic cytopenia, n=115). In healthy subjects, the 95%-percentile ranged between 2.4 and 9.5 ng/ml (median 5.7 ng/ml). Healthy controls aged ≤ 16 years had a slightly lower median serum tryptase level compared to older control subjects (p=0.03). Among pts with non hematologic disorders, slightly elevated serum tryptase levels (up to 24 ng/ml) were detectable in 6/18 pts with severe renal failure and in 3/29 pts with helminth infections. When analyzing hematologic malignancies, tryptase levels >15 ng/ml could be detected in pts with myeloid neoplasms exclusively, whereas in most pts with lymphoproliferative disorders, tryptase levels were normal. Among myeloid neoplasms, elevated tryptase levels were recorded in 83% of the pts with SM, 36% of pts with AML, 34% of the pts with CML, and 29% of the pts with MDS. The highest tryptase levels, sometimes exceeding 1000 ng/ml, were found in pts with SM or AML-M4eo. In pts with CML, elevated tryptase levels were found to be associated with an unfavorable prognosis concerning survival (survival in CML pts with tryptase <15 ng/ml: 100% vs 71% in those with >15 ng/ml, after 60 months, p<0.007). In most pts with AML, SM, or CML, the initially enhanced serum tryptase levels decreased in response to successful cytoreductive or targeted therapy. In pts with AML, a persistently elevated serum tryptase in complete hematologic remission (CR) was found to be associated with an increased risk of relapse compared to those with normal tryptase in CR. In summary, tryptase is a new and simple diagnostic marker of myeloid neoplasms. In SM, AML, and CML, tryptase is also a useful marker to monitor (minimal residual) disease during treatment with cytoreductive or targeted drugs. Moreover, in AML and CML, measurement of tryptase is of prognostic significance. All in all, tryptase is recommended as a new important serum-marker in clinical hematology.
Artificial Neural Network Analysis of HOX Gene Expression Profiles Predicts Prognostic Subgroups in Acute Myeloid Leukemia.
Authors: Finnegan Damian P.J. , Quinn Michael F. , Humphreys Mervyn , Lappin Terence R.J. , McMullin Mary Frances , Thompson Alexander , Ball Graham R.
Published: October 2019

The acute myeloid leukemias (AMLs) are a heterogeneous group of hematological malignancies with diverse clinical outcomes. Pre-treatment karyotype analysis identifies biologically distinct subgroups and is currently used as a predictor of response to induction chemotherapy and risk of relapse. Cases may be stratified into one of three prognostic groups as follows: relatively favorable prognosis [t(8;21), t(15;17) and inv(16)]; adverse prognosis [−5/del(5q), −7, abnormalities of chromosome 3q and complex karyotype]; and intermediate prognosis [remainder including normal karyotype]. HOX genes encode master transcription factors which regulate key developmental processes including differentiation, proliferation and apoptosis. Humans have 39 HOX genes and multiple lines of evidence implicate their deregulated expression in the pathogenesis of AML. Drabkin et al. (Leukemia2002; 16: 186–95) have reported that AMLs with a relatively favorable prognostic karyotype are associated with low levels of HOX gene expression whereas AMLs with an adverse prognostic karyotype have higher levels of expression. To further characterize HOX gene expression in cytogenetic prognostic groups we determined the expression profiles of 26 HOX genes by real-time quantitative PCR (Q-PCR) in diagnostic samples, representative of the three prognostic groups, from 26 patients with de novo AML. Profiles were then analyzed using Artificial Neural Network based computational approaches to identify a subset of HOX genes which could discriminate between prognostic groups in a predictive fashion. Predictive models were developed for each prognostic group. Predictive classification performance for prognostic groups based on blind data of 88%, 92%, and 97% (with equal sensitivity and specificity) were achieved for the three prognostic groups. The models were interrogated to determine the nature of the relationship between the key HOX genes identified and prognostic group. The relatively favorable prognosis group was primarily defined by downregulation of HOXA5 and upregulation of HOXC4. The intermediate prognosis group was characterized by upregulation of HOXB3 and downregulation of HOXD10 and the adverse prognosis group by downregulation of both HOXC5 and HOXD3. Although the sample size is small, the results show that Artificial Neural Network based computational approaches are capable of further characterizing HOX gene expression within AML prognostic groups as determined by presenting karyotype and that measuring the expression levels of a small number of HOX genes at diagnosis can provide useful clinical information in cases where karyotype analysis has been unsuccessful.
Prognostic Implications of the Immunophenotype in Biphenotypic Acute Leukemia.
Authors: Lee Je-Hwan , Min Yoo-Hong , Chung Chul Won , Kim Byoung Kook , Yoon Hwi-Joong , Jo Deog-Yeon , Shin Ho-Jin , Bang Soo-Mee , Won Jong-Ho , Zang Dae Young , Kim Hyeoung Joon , Chi Hyun-Sook , Lee Kyoo Hyung , Cheong June-Won , Kim Jin Seok , Kim Sun-Hee , Park Seonyang , Park Su Yon , Chung Jooseop , Lee Jae-Hoon , Park Chan-Jeoung
Published: October 2019

Biphenotypic acute leukemia (BAL) is a rare yet defined type of acute leukemia. We investigated the incidence, clinicopathologic characteristics, and clinical outcomes of BAL in Korean adults. The present study retrospectively analyzed clinicopathological and clinical data from 43 adult patients with BAL, defined using the EGIL scoring system, from 11 Korean institutes. The incidence of BAL was 2.1% among acute leukemias; 2.3% in male and 1.9% in female. Median age of 43 BAL patients, 25 males and 18 females, was 38 years (range, 16–74). The immunophenotype was myeloid/B-lymphoid (M+B) in 31 (72.1%), myeloid/T-lymphoid (M+T) in 10 (23.3%), and B/T-lymphoid (B+T) and myeloid/B/T-lymphoid (M+B+T) in one (2.3%) each. 37 patients had the results of cytogenetic analyses and Ph chromosome (n=14, 37.8%) was the single most common abnormal finding. Intensive induction chemotherapy was given in 36 of 43 patients: AML-type in 13 (36.1%), ALL-type in 8 (22.2%), and combined type in 15 (41.7%). Complete remission (CR) was induced in 29 patients (80.6%). The CR rate was significantly lower in M+T (5 of 9, 55.6%) than M+B (22 of 25, 88.0%) (P=0.039). Other unfavorable factors for CR were absence of CD19 (P=0.029) or CD20 (P=0.046), and presence of CD2 (P=0.040). The CR rates were not significantly different according to cytogenetic finding or type of induction chemotherapy. After median follow-up duration of 712 days among surviving patients, 11 patients relapsed with median relapse-free survival (RFS) of 3.08 years and 4-y RFS of 38.3% and 18 patients died with median overall survival (OS) of 2.49 years and 4-y OS of 30.1%. Multivariate analysis showed that high leukocyte counts (≥ 30,000/μl) at diagnosis (OR, 5.922; 95% CI, 1.379–25.429; P=0.017) was an independent unfavorable prognostic factor for RFS, and high leukocyte counts at diagnosis (OR, 9.348; 95% CI, 3.014–28.993; P<0.001) and M+T phenotype (OR, 3.259; 95% CI, 1.136–9.344; P=0.028) were independent unfavorable prognostic factors for OS. In summary, BAL was found in 2.1% of adult acute leukemias and M+T phenotype showed significantly poorer response to induction chemotherapy and inferior overall survival compared to M+B phenotype.
Hypomethylation Induction and Molecular Response after Decitabine Therapy in Chronic Myelomonocytic Leukemia (CMML).
Authors: Oki Yasuhiro , Jelinek Jaroslav , Kantarjian Hagop M. , Issa Jean-Pierre J.
Published: October 2019

Decitabine has shown therapeutic activity in patients with MDS and CMML. The mechanisms of response to therapy remain incompletely understood. In particular, the relative contribution of this drug’s ability to induce hypomethylation and cytotoxicity remains unclear. To address this issue, we studied the dynamics of neoplastic cell clearance during decitabine treatment determined by quantitative monitoring of the mutant allele using pyrosequencing. DNA extracted from peripheral blood mononuclear cells from consented patients with CMML in a decitabine phase II study were first screened for JAK2 and NPM1 mutations as previously reported. We identified three patients with mutations (two with JAK2 mutation, one with NPM1 mutation) and samples at multiple points during therapy were available. All three carried normal karyotype. LINE repetitive element methylation and several other gene specific methylations were also assessed. In the three patients, LINE methylation decreased after each cycle of therapy, and recovered to near baseline after the drug was stopped (e.g. during the first cycle, average relative hypomethylation from baseline was 13.9% at day 12 and 6.5% at day 28). At the same time, the proportion of circulating neoplastic cells decreased slowly after the first cycle (decrease by 19.3% at day 12 and 13.5% at day 28). A substantial decrease in mutant allele percentage was observed after cycles 2, 3, and 2 in patients 1, 2, and 3, respectively. Clinical complete responses were achieved along with molecular responses at cycles 5, 4 and 2, respectively. Patients 1 and 2 showed complete disappearance of detectable neoplastic clones, and had sustained remissions (duration 1.5 and 2.5 years). In patient 3, the proportion of neoplastic cells was lower than baseline but still detectable at clinical remission, and the remission only lasted 8 months. We conclude that neoplastic cell clearance after decitabine therapy in CMML is observed after several courses of therapy, and is initially seen concurrently with hypomethylation. While LINE methylation returns to its steady state values after completion of decitabine infusion, the tumor elimination process slowly continues. Our data suggest a non-cytotoxic mechanism of action for the drug, whereby the biology of the neoplastic clone is altered by hypomethylation, leading to delayed clearances of unknown mechanism. Possibilities include an immune response and effects on the neoplastic (or normal) stem cells.

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