Figure 1. Unsupervised hierarchical clustering of gene expression data from bone marrow samples of 31 children with acutelymphoblasticleukemia. Cluster dendrogram based on 333 fil- tered probe sets (see methods for filter criteria). The upper main branch includes precursor B cell leukemias, whereas the lowest branch identifies two T-cell leukemias. The left column shows the patient number, cytogenetic characterization is listed in the middle column, risk group assignment is pre- sented in the right column. The dendro- gram did not change substantially when more (or even all) probe sets were used (results not shown). *normal indicates DNA index of 1 and no TEL-AML1, BCR- ABL, E2A-PBX1 or MLL-AF4 rearrange- ment.
38 Li Benshang, Brady Samuel W, Ma Xiaotu, Shen Shuhong, Zhang Yingchi, Li Yongjin, Szlachta Karol, Dong Li, Liu Yu, Yang Fan, Wang Ningling, Flasch Diane A, Myers Matthew A, Mulder Heather L, Ding Lixia, Liu Yanling, Tian Liqing, Hagiwara Kohei, Xu Ke, Zhou Xin, Sioson Edgar, Wang Tianyi, Yang Liu, Zhao Jie, Zhang Hui, Shao Ying, Sun Hongye, Sun Lele, Cai Jiaoyang, Sun Hui-ying, Lin Ting-Nien, Du Lijuan, Li Hui, Rusch Michael, Edmonson Michael N, Easton John, Zhu Xiaofan, Zhang Jingliao, Cheng Cheng, Raphael Benjamin R, Tang Jingyan, Downing James R, Alexandrov Ludmil B, Zhou Bin-Bing S, Pui Ching-Hon, Yang Jun J, Zhang Jinghui. Therapy-induced mutations drive the genomic landscape of relapsed acutelymphoblasticleukemia. Blood 2019;135:41–55. doi:10.1182/blood.2019002220.
Integrins, a form of cell adhesion receptors, are highly expressed in leukemia cells, including integrin α4 and α6 [ 18 , 19 ]. Our previous preclinical studies demonstrated the successful eradication of primary B-cell acutelymphoblasticleukemia (ALL) cells engrafted in NSG (NOD. Cg-Prkdc scid Il2rg tm1Wjl /SzJ) mice by treatment with chemotherapeutic drugs combined with integrin blockade [ 18 , 19 ]. To characterize the physical properties of the chemotherapeutics-treated B-ALL cells, we used acoustic tweezers to measure the deformable capacity, which was found to be increased by the chemotherapeutic drug combination [ 18 ]. Furthermore, compared with its isotype control (IgG1), the antibody against α 6 (P5G10) but not Tysabri (humanized antibody against α4) markedly reduced the deformability [ 19 ]. However, the role of the motility of chemotherapeutics-treated ALL cells has not yet been widely explored. In the present study, the primary B-ALL cells, LAX7R, LAX56, and ICN24, were monitored using time-lapse cinematography for 12 h to investigate their motility in vitro.
Acutelymphoblasticleukemia is a heterogeneous disease with important prognos- tic subtypes . The diagnosis of ALL is based on the presence of 25 % or more blasts in bone marrow (BM). The French-American-British (FAB) classification al- lows distinction of three subgroups according to the size and morphology of the blasts (L1 to L3) . However, other methods have become essential for leukemia classification in addition to the microscopic assessment of leukemic blasts [94,142]. Flow cytometric analysis of bone marrow or peripheral blood with a panel of antibodies against surface and cytoplasmatic markers is required to distinguish relevant immunophenotypes (B-cell precursor (BCP), mature B-cell or T-cell ALL). Cytogentic analysis like fluorescene in situ hybridization (FISH) can also be used to detect chromosomal abnormalities. Additionally, gene expression profiling (GEP) allows separation of new subtypes opposing favorable and unfavorable out- come on an experimental level [29,94,97,142,150]. The importance of genetic ab- normalities of ALL cells is reflected in the classification of acute leukemias of the World Health Organisation (WHO), composed in Table 1 .
The aim of this study was to assess the expression of protein tyrosine kinases (PTK) in acutelymphoblasticleukemia (ALL) patient samples at the protein level. Xenotransplanted primary ALL blasts and ALL cell lines were used as model system for the functional analysis of the role of PTK in ALL. The analysis revealed that Lyn, a member of the Src family kinase (SFK), was prominently expressed in a subgroup of ALL patient samples. To further investigate the biological consequence of elevated Lyn expression in ALL cells, Nalm6 and CALL3 cells were used as a model which recapitulated the high and low Lyn expression profile observed in patient specimens, respectively. Lyn is known to be associated with pre-BCR after receptor crosslinking. Analysis of the functional role of Lyn upon shRNA mediated Lyn repression and pre-BCR crosslinking showed that phosphorylation of downstream signaling proteins was strikingly reduced or delayed in Nalm6 cells. In addition, cell proliferation was substantially reduced in Nalm6 Lyn-knockdown cells. Conversely, an increase in the tyrosine phosphorylation was found in CALL3 Lyn-knockdown cells. Membrane microdomain, called lipid rafts, were shown to concentrate and regulate SFK 111 . However, data of the Lyn localization in the plasma membrane indicates that, whereas Lyn was exclusively present within defined lipid rafts in CALL3 cells, the protein was aberrantly localized all over the membrane in Nalm6 cells. The Lyn mislocalization was likely independent of lipid rafts and it could enable Lyn to interact with other proteins located outside rafts structures and promote its activation. Ultimately, preliminary data suggests that overexpression of Lyn is implicated in resistance to tyrosine kinase inhibitor (TKI) treatment.
In children and adolescents the most frequent malignant disorder is acutelymphoblasticleukemia (ALL) with 80% cure rates, largely due to the improvement of multiagent chemotherapy regimen although 20% of patients encounter relapse (Conter, Arico et al. 2010, Pui and Evans 2006, Pui and Jeha 2007, Schrappe, Nachman et al. 2010, Schrappe, Reiter et al. 2000). Most of the conventional drugs used to treat ALL either target DNA, or inhibit nucleic acid synthesis, or interfere with the mitotic spindle formation and possess a narrow therapeutic index (Pui and Jeha 2007). On the other hand, the major cause of treatment failure is relapse for which a number of causes have been identified and inadequate therapy is the main cause (Pui and Evans 2006, Schrappe, Hunger et al. 2012). This dismal treatment outcome warrants for alternative strategies for the early identification of high-risk ALL as well as for therapies targeting aberrant signal transduction pathways. Previously, it has been identified that in vivo proliferation of patient leukemia cells transplanted onto NOD/SCID mice is indicative of poor patient outcome and characterized by a specific transcript profile involving survival pathways (Meyer, Eckhoff et al. 2011). The present study addresses activity of mTOR, a key survival pathway, in xenograft ALL cells on a functional level including preclinical evaluation of therapy targeting this key cellular pathway.
higher in the group treated with E. coli asparaginase than in that treated with ERW-asparag- inase. Similar results were found in a study by Woo et al. 8 A different trend has been found in studies performed so far in patients treated with PEG-asparaginase. Vieira Pinheiro et al. 30 studied patients treated with PEG-asparag- inase within the German Cooperative AcuteLymphoblasticLeukemia (COALL) study and Rizzari et al. 15 patients treated with the same product within the AIEOP ALL 2000 study. Overall, both studies showed that CSF asparagine levels in patients treated with PEG- asparaginase were undetectable (i.e., below the detection limit) only in a fraction of patients, no matter if serum asparaginase activity levels were much higher than 100 IU/L. Additional studies reported by the Nordic Society of Pediatric Hematology and Oncology and even more recently by the Dutch Childhood Leukemia Study Group (DCLSG) confirmed these observations. 14,27,31,32
Figure 12. The retinoic acid inducible gene I (RIG-I)/ melanoma differentiation associated gene-5(MDA- 5)/ mitochondrial antiviral signaling (MAVS)/ interferon-β (IFN-β) axis is defective in ALL cell lines but intact in B-cell precursor acutelymphoblasticleukemia (ALL) xenograft cells T and B-cells isolated from peripheral blood mononuclear cells (PBMC) of 5 and 6 donors, respectively, ALL cell lines and B-cell precursor ALL xenografts #6, #13, #15 and #19 were infected with measles virus Edmonston (MV-Edm) at a multiplicity of infection (MOI) of 1 or were not infected. Messenger ribonucleic acid (mRNA) was isolated 12 hours (h) later. mRNA expression of RIG-I, MDA-5, MAVS, interferon response factor-3 (IRF-3), IRF-1, IFN-β, interferon-α/β receptor (IFNAR) 1, IFNAR2, interferon- stimulated gene-15 (ISG-15) and interferon-induced protein with tetratricopeptide repeats 1 (IFIT1) was measured by quantitative reverse transcriptase (qRT)- polymerase chain reaction (PCR) and is shown relative to expression of β-ACTIN. Statistical analysis by 1-way ANOVA for IFN-β: T-cells vs T-ALL cell lines p < 0.05, B-cells vs B-ALL cell lines and vs xenografts p < 0.001, B-ALL cell lines vs xenografts p < 0.001; for RIG-I: T-cells vs T-ALL cell lines p < 0.05, B-cells vs B-ALL cell lines and vs xenografts, and B-ALL cell lines vs xenografts all p < 0.001; for MDA-5: T-cells vs T- ALL cell lines, B-cells vs B-ALL cell lines and vs xenografts, and B-ALL cell lines vs xenografts all p < 0.001. This figure has previously been published in . With permission of the Ferrata Storti Foundation.
treatment initiation phase by analyzing white blood cell counts (prednisone response = PR) (Dordelmann et al. 1999). ALL treatment performed according to the AEIOP-BFM ALL 2000 clinical trial (Associazione Italiana Ematologia ed Oncologia Pediatrica – Berlin Frankfurt Münster – AcuteLymphoblasticLeukemia 2000), comprises risk stratification classifying patients into three different groups – standard risk (SR), medium risk (MR) and high risk (HR), based on their prednisone response and the occurrence of translocations t(4;11) and t(9;22) (Conter et al. 2010). Additionally, minimal residual disease (MRD) is defined by the amount of remaining ALL cells detected on submicroscopic level by PCR analyzing IgG/TCR rearrangements and is estimated for every patient (van Dongen et al. 1998). But even risk stratification according to MRD is not sufficient to securely detect patients with a high risk for relapse, which would allow to intensify therapy already during the initial phase of their treatment (Conter et al. 2010; Schrappe et al. 2000). Therefore, new diagnostic and therapeutic approaches are necessary to understand the underlying altered biology and pathobiology of pediatric ALL in order to allow the identification of high risk and early-relapse patients.
An estimate of 6000 US citizens develop acutelymphoblasticleukemia (ALL) every year (Siegel et al, 2012). In 80% of these cases the leukemia cells express B lymphocyte markers alongside early progenitor antigens (Armstrong, 2005), consistent with the malignant transformation of conventional precursor B lymphocytes during their maturation in the bone marrow (Zhou et al, 2012). Over the last 50 years, the introduction of combination chemotherapy, improved risk stratification and enhanced supportive care led to a steady increase in the survival rates of ALL patients. Today, state of the art therapy cures an average of 85% of childhood and 40% of adult ALL cases (Pui et al, 2009; Hunger et al, 2012; Sive et al, 2012). However, ALL treatment remains challenging. The extensive exposure of children to cytotoxic agents causes side effects including cognitive impairment and second malignancies even years after completing therapy (Conklin et al, 2012; Mody et al, 2008; Nottage et al, 2011). Moreover, despite strong efforts 60% of adults still succumb to their disease. It is widely accepted that further improvements of ALL therapy will have to rely on novel more targeted treatment approaches. This includes the specific inhibition of kinases that promote the clonal expansion of B-ALL cells, using small molecule kinase inhibitors. However, the successful application of such targeted agents requires a profound knowledge of the biological mechanisms driving malignant proliferation.
To the Editor
As one of the leading internet information platforms (EMF-Portal at RWTH Aachen University www.emf- portal.org) we systematically screen and summarize scientific research on the effects of electromagnetic fields (EMF). Thus we came across the publications by Tabrizi and Bidgoli (2015) and Tabrizi and Hosseini (2015) on prenatal and postnatal exposure to high voltage power lines and the risk of childhood acutelymphoblasticleukemia (ALL), both published in your journal in April and December 2015, respectively.
As one of the leading internet information platforms (EMF-Portal at RWTH Aachen University www.emf- portal.org) we systematically screen and summarize VFLHQWLÀFUHVHDUFKRQWKHHIIHFWVRIHOHFWURPDJQHWLFÀHOGV (EMF). Thus we came across the publications by Tabrizi and Bidgoli (2015) and Tabrizi and Hosseini (2015) on prenatal and postnatal exposure to high voltage power lines and the risk of childhood acutelymphoblasticleukemia (ALL), both published in your journal in April and December 2015, respectively.
childhood acutelymphoblasticleukemia. We prospectively com- pared the efficacy and safety of antithrombotic interventions in the consecutive leukemia trials ALL-BFM 2000 and AIEOP-BFM ALL 2009. Patients with newly diagnosed acutelymphoblasticleukemia (n=949, age 1 to 18 years) were randomized to receive low-dose unfractionated heparin, prophylactic low molecular weight heparin (enoxaparin) or activity-adapt- ed antithrombin throughout induction therapy. The primary objective of the study was to determine whether enoxaparin or antithrombin reduces the incidence of thromboembolism as compared to unfractionated heparin. The principal safety outcome was hemorrhage; leukemia outcome was a secondary endpoint. Thromboembolism occurred in 42 patients (4.4%). Patients assigned to unfractionated heparin had a higher risk of throm- boembolism (8.0%) compared with those randomized to enoxaparin (3.5%; P=0.011) or antithrombin (1.9%; P<0.001). The proportion of patients who refused antithrombotic treatment as allocated was 3% in the unfractionated heparin or antithrombin arms, and 33% in the enoxaparin arm. Major hemorrhage occurred in eight patients (no differences between the groups). The 5-year event-free survival was 80.9±2.2% among patients THROMBOTECT – a randomized study
to childhood acutelymphoblasticleukemia treatment. To eluci- date genetic predisposition and asparaginase-associated pancre- atitis pathogenesis, ten trial groups contributed remission samples from patients aged 1.0−17.9 years treated for acutelymphoblasticleukemia between 2000 and 2016. Cases (n=244) were defined by the presence of at least two of the following criteria: (i) abdominal pain; (ii) levels of pancreatic enzymes ≥3 x upper normal limit; and (iii) imaging compatible with pancreatitis. Controls (n=1320) completed intended asparaginase therapy, with 78% receiving ≥8 injections of pegylated-asparaginase, without developing asparaginase-associated pancreatitis. rs62228256 on 20q13.2 showed the strongest association with the development of asparaginase-associated pancreatitis (odds ratio=3.75; P=5.2x10 -8 ). Moreover, rs13228878 (OR=0.61; P=7.1x10 -6 ) and rs10273639 (OR=0.62; P=1.1x10 -5 ) on 7q34 showed significant association with the risk of asparaginase-associated pancreatitis. A Dana Farber Cancer Institute ALL Consortium cohort consisting of
Figure 6: Induction of transcripiton of Toll-like receptor 9 (TLR9) messengerRNA (ri- bonucleic acid) in B cell precursor-acutelymphoblasticleukemia cells (BCP- ALL cells) through treatment with interleukin-4 (IL-4) and cluster of dier- entiation (CD) 40 Ligand (CD40L). Reverse transcription polymerase chain reaction (PCR) was done on the three BCP-ALL cell lines 018Z, RS4;11 and KOPN-8 for quan- titative analysis. After 48 hours of incubation with IL-4 (400 U/ml), CD40L (1 µg/ml) along with an enhancer for ligands (1 µg/ml) plus or minus cytosine-phosphate-guanosine oligodeoxynucleotide B (CpG B) (2.5 µg/ml) total RNA was extracted from 2x10 6 BCP- ALL cells per sample. As control served untreated cells of the same cell line. PCR signals were normalized to average expression of the housekeeping genes TATTA box-binding pro- tein (TBP) and RPL-32 for each sample (∆CT). To compare each sample to the untreated control, ∆∆CT values were calculated by subtraction of ∆CT values of untreated from treated samples. Final values were plotted as 2 −∆∆CT . Data results from three (018Z), six (RS4;11) and four (KOPN-8) independent experiments. Error bars indicate standard error of means. * indicates p < 0.05, ** indicates p< 0.01.
MEIS2, a transcription factor encoded by MEIS2, a homeobox gene, is known to play as a crucial role in differentiation during embryogenesis, development of limbs and various organs. Our group could recently show that the co-expression of MEIS2 with AML-ETO (Acute Myeloid Leukemia 1/Eight-Twenty One oncoprotein) can induce acute myeloid leukemia in mice. But there are scarce data about MEIS2 involvement in acutelymphoblasticleukemia (ALL). In this study, we aimed to characterize MEIS2 in acutelymphoblasticleukemia. We could show that MEIS2 is aberrantly expressed in bone marrow samples of patients with acutelymphoblasticleukemia in comparison to healthy controls. In addition, we demonstrated that MEIS2 is significantly higher expressed in pediatric samples than adult ALL samples. A methylation analysis of two CpG islands showed that in eight of nine patients MEIS2 expression inversely correlated to the extent of promotor methylation. Knockdown of MEIS2 in acutelymphoblasticleukemia cell lines Tanoue (B-ALL) and Jurkat (T-ALL) showed a significant proliferation inhibition and reduced clonogenic capacity. Flow cytometric analysis of knockdown cells did not show marked differences in apoptosis, however, cell cycle assays revealed a cell cycle arrest in the G0-phase following MEIS2 knockdown, suggesting a role of MEIS2 in cell cycle progression/regulation. RNAseq after knockdown of MEIS2, resulted in significantly differentially expressed genes belonging to pathways involved in B- and T- cell activation, transcription regulation by bZIP transcription factor, EGF- FGF-, TGF-beta, VEGF-signaling as well as RAS- and p38- MAPK pathway. Genes like RAC2 and CD74 showed a marked downregulation and genes like CREB5 and CTTN were upregulated following MEIS2-knockdown, which was further validated by qRT-PCR.
federal states, representing a total base population of 33 million people, were included (Table 1). Patients age 15 or older with a primary diagnosis of ALL (ICD-10 code C91.0) in 1997–2006 and with mortality follow up through December, 2006 were included. Cancer topography, morphology, and behavior were originally coded in accordance with the International Classification of Disease for Oncology (ICD-O)-3 guidelines and later converted into ICD-10 using ‘IARCcrgTools’ . Patients with both B-cell and T-cell acutelymphoblasticleukemia are covered under this diagnostic code in ICD-10. For some registries, data were available starting from later years only. Cases both with and without preceding cancers were included. Because there were data quality issues for patients age 70+ in some of the German registries, only data for patients age 15–69 were included. In order to compare population level survival for ALL in Germany with survival in the United States (US), data from the Surveillance, Epidemiology, and End Results (SEER13) database were analyzed . The same inclusion criteria as for patients from the German cancer registries were applied for the same time period. The SEER13 database includes data from 13 regional cancer centers in the US, covering a population of about 39 million people. Centers are chosen for inclusion based on their high quality and epidemiologically interesting population groups. The SEER population is considered to be similar to the general US population with respect to most sociodemographic characteristics , although it may be more affluent than average and may have slightly higher than average survival for some cancers .
Over the past decades, long-term survival rates in childhood malignancies and especially in acutelymphoblasticleukemia (ALL) have increased substantially with improved and adapted treatment regimens ( 1 – 3 ). In the growing group of survivors, late effects from cancer and its treatment are an increasing burden compromising health and quality of life. Endocrine late effects in general play a major role in survivorship morbidity ( 4 – 7 ). Bone health has been shown to be impaired in a significant number of children and adults surviving pediatric malignancies ( 8 ). Mostoufi-Moab and Halton review the subject for patients with childhood ALL ( 9 ). At time of diagnosis there is an incidence of vertebral fractures of up to 16% in the pediatric age group ( 10 ) and 72% develop osteonecroses, which often are asymptomatic ( 11 ). Patients undergoing treatment for ALL are particularly affected by low bone mineral content and low bone mineral density (BMD) ( 12 , 13 ). Some studies report this to persist until adulthood ( 14 , 15 ) while others report a normalization over time, especially in patients without cranial irradiation ( 16 ).
would comprise less LSCs able to home in mice than 1 x 10 6 cells of ALL-265. Correspondingly, some publications could show that in B-ALL many cells exhibit leukemia propagating activity whereas in AML stem cell frequencies tend to be markedly lower (Kelly et al., 2007; Rehe et al., 2013; Sarry et al., 2011). In fact, for ALL-265, we determined a very high LSC frequency of 1 stem cell in 27 cells using limiting dilution transplantation assay. However, we did not clearly define the stem cell frequencies of AML-393 and AML-346. For AML-393, the amount of stem cells in the sample has to be at least 1 in 2,000 cells since all of the mice that had been transplanted with 2 x 10 3 cells developed leukemia, but it may be even higher. Consequently, more experiments would be needed to prove this hypothesis. Still, our findings may suggest that ALL and AML samples differ in the amount of LSCs but more experiments would be required to strengthen this idea. In summary, these findings indicate that RGB marking combined with xenotransplantation enabled visualization of engraftment of leukemia propagating cells in mice. By transplanting limiting numbers of RGB ALL-265 and RGB AML-393 cells, we discovered that the amount of differently colored populations after the additional mouse passage was dependent on the amount of transplanted cells as expected. Transplantation of many cells caused only small changes in expressed colors whereas upon transplantation of few cells, we observed a prominent decrease of colors. Ultimately, transplantation of only some hundred cells resulted in engraftment of single cells and in generation of single cell clones.