Anaplastic large cell lymphoma (ALCL) is a distinct form of non-Hodgkin lymphoma that accounts for 10-15% of all childhood lymphomas.Differently from ALCL in adults, where about 30% of cases are ALK+, ALCL in children is nearly universally ALK+, and in almost all of the cases, it is characterized by the t(2;5)(p23;q35) translocation, which results in the expression of the hybrid oncogenic tyrosine kinase NPM-ALK.NPM-ALK fusion protein is responsible for ALCL development, and throughout its constitutive tyrosine kinase activity, it regulates signalling pathways critical for proliferation, cell-cycle control and survival of ALCL cells.So far, few studies have investigated ALCL gene expression profiling, and most of them have used heterogeneous populations of paediatric and adult cases.Main aim of such studies was to identify whether ALK+ and ALK- ALCL had a different transcriptional program, and although differences were found, similarities were also detected.Based on the fact that paediatric ALCL are almost exclusively ALK+, our study focussed on investigating the potential heterogeneity inside ALK+ ALCL rather than differences between ALK+ and ALK- tumors.In order to investigate the transcriptional characteristics of ALK+ ALCL, we performed a global microarray-based transcriptional anal. on 23 paediatric tumor samples, 12 reactive lymph nodes and 4 ALCL cell lines (3 ALK+ and 1 ALK-) (Supplementary Materials).As previously reported, we found that ALK+ tumors displayed downregulation of both B-cell and T-cell receptor signalling mols. (such as ARNT2, CD3G, CD24, CD27, CD40, CD79A, NFATC2, PIK3CD, TNFRSF10A) in addition to T helper cell surface mols. (such as CD19, CD28, CTLA4), (Supplementary Table S1), consistent with the notion that this malignancy originates from undifferentiated normal CD4+ T lymphocytes.5 To explore the mol. differences between clin. homogeneous ALK+ ALCL, we performed unsupervised clustering anal. of the tumor samples, identifying two clearly distinct subgroups of patients.Indeed, supervised anal. identified two different gene expression signatures, both related to the diverse amount of NPM-ALK in ALCL cells (ALK-low and -high).Of note, ALCL cell lines clustered together and had a gene expression signatures more similar to ALK-high than to ALK-low samples (Figure 1a; Supplementary Table S2).The ALK-high/low signatures were not affected by sample or patient selection bias, as our novel ALK group classification was independent of the lymph node infiltration rate, as the percentage of ALK+ cells was about 40% in the large majority of cases and it could be applied to indipendent ALCL studies (Supplementary Figure S1).NPM-ALK levels were assessed by real-time quant. reverse transcriptase PCR (RQ-PCR) in the same set of patients showing a significant correlation with gene expression profiling data (Pearson's correlation coefficient 0.79, P<0.0001) (Figures 1b and c, resp.).Addnl. 19 paediatric ALCL cases were tested by RQ-PCR, and overall the full data set, including 42 samples, displayed a bimodal distribution for NPM-ALK expression consistent with the presence of an ALK-low and an ALK-high group.The median expression values were 2.500 NPM-ALK copies/10.000 ABL in ALK-low cases and 125.000 NPM-ALK copies/10.000 ABL in ALK-high patients (Welch's t-test, P<0.0001 Figure 1d).In addition, when NPM-ALK protein expression was measured in a subset of our cases, selected based on tissue availability (Figure 1e), we found that expression and phosphorylation of NPM-ALK kinase correlated with mRNA levels of the two groups (Figure 1f), supporting the concept that ALK+ ALCL can be distinguished based on NPM-ALK endogenous expression level.We did not observe significant differences between the two subgroups based on clin. characteristics, although patients with central nervous system involvement (n=4) belonged to the ALK-high subgroup (Supplementary Table S3).Interestingly, 9 out of the 12 patients who experienced relapse were ALK-high cases and had a median time of relapse of 5 mo compared with the median time of relapse of 30 mo of the 3 ALK-low relapsed patients.Gene set enrichment anal. using the MSig DB database (Mol. Signature Database, Oncogene signature C6, http://www.broad.mit.edu.libproxy1.nus.edu.sg/gsea/) showed a pos. enrichment for genes involved in the interleukin (IL) signalling pathway (specifically IL-2, IL-15 and IL-21) in ALK-low compared with ALK-high ALCL samples, while genes set associated with cell growth and division (CyclinD1, MYC and E2F1) were significantly upregulated in ALK-high cases (Figure 1g).The IL-2 signalling pathway is critical for CD4+ T-cell maturation and function,6, 7 and its downregulation occurs in ALCL cell lines in response to NPM-ALK expression and activity.Consistently, we showed that IL-2Rγ was expressed in ALK- (FE-PD) ALCL cell line but not in ALK+ (KARPAS-299, SUD-HL-1 and SUP-M2) cell lines (Figure 2a).In order to evaluate the IL-2Rγ expression in ALCL primary tumor cases, we analyzed, by flow cytometry, nine patients (four ALK-low and five ALK-high), for whom tissue samples were available.The results showed that tumor cells in ALK-low patients ubiquitously expressed IL-2Rγ (ALK+/IL-2Rγ+ cells >95%), whereas ALK-high patients displayed a more heterogeneous pattern of expression (Figures 2b and c).Taking into account that ALK-high patients relapsed more frequently and rapidly than ALK-low patients, it seems that in ALK-low cases NPM-ALK is unable to make transformed lymphocytes completely 'deaf' to ILs, and IL signalling retention, in turn, contributes to a less aggressive tumor phenotype.Functional annotation anal. performed by gene set enrichment anal. in ALK-high tumor samples revealed increased expression of genes involved in cell growth and survival, such as cyclins and Aurora kinases A (AURKA) and B (AURKB).These genes are known not only to affect cell proliferation but also regulate chromosomes' segregation and mitosis.Hence, to validate our bioinformatics anal., we took advantage of com. available Aurora kinase inhibitors, which were used to treat a panel of ALK+ ALCL cell lines (KARPAS-299, SU-DHL-1 and SUP-M2) representative of the ALK-high subgroup.As expected, marked growth inhibition of ALCL cells was observed upon administration of AURKA and AURKB inhibitors Alisertib (MLN8237) and Barasertib (AZD1152), resp., and this was associated with cell-cycle arrest and induction of apoptosis (Figure 2d).In particular, the exposure of ALCL cells to increasing concentrations of MLN8237 and/or AZD1152 led to time-dependent inhibition of cell growth, which, consistent with Aurora kinases' involvement in mitotic entry and cytokinesis, was characterized by a dose-dependent 'growth plateau' typical of a cytostatic cell response (Figure 2d).In contrast, Aurora kinases inhibitors did not cause changes in healthy peripheral blood mononuclear cells' proliferation (stimulated with or without phytohaemagglutinin), when used in the same exptl. conditions (Figure 2e).Previous data have shown that Aurora inhibitors exert their activity leading to accumulation of polyploid cells with an endoreplication phenotype before mitotic catastrophe or apoptosis occur.Indeed, when drug-inhibitory effects were investigated in more detail, we found that both compounds, administered for 48 h as single agents or in combination, led to a dramatic increase of the polyploid cell content (8N) over the diploid (2N) and tetraploid (4N) cell population (Supplementary Figure S2A), whereas at earlier time points (24 h) they induced a provisional arrest in G2/M phase and accumulation of tetraploid cells only (data not shown).Prolonged drug exposure, instead, affected cell survival, as the fraction of the cells undergoing apoptosis (Annexin V-pos., AV+) or necrosis (Propidium Iodide pos., PI+) increased over time, after 72 h of treatment (Supplementary Figure S2B).AURKA and AURKB have been recently detected in non-Hodgkin lymphoma; however, their involvement in ALCL tumorigenesis, as well as their importance as therapeutic targets, has not been investigated.Herein we provide evidence that AURKA and AURKB may be considered novel drug targets in ALCL and that Aurora kinase inhibitors are effective agents in preclin. ALCL models.Their efficacy depends on their general mode of action and results in inhibiting proliferation, survival and cell-cycle progression of ALCL cells.Overall, our data suggest that the heterogeneous levels of aggressiveness observed in ALCL tumors may be explained by a NPM-ALK distinct gene expression and transcriptional program.High-level ALK+ cells seems to acquire a selective growth advantage compared with ALK-low cells.This concept merits attention to exploit novel therapeutic strategies in this malignancy that may increase curability and survival of children with ALCL.
