Patients diagnosed with Anaplastic Large Cell Lymphoma (ALCL) are still treated with toxic multi-agent chemotherapy and as many as 25-50% of patients relapse. To understand disease pathology and to uncover novel targets for therapy, Whole-Exome Sequencing (WES) of Anaplastic Lymphoma Kinase (ALK)+ ALCL was performed as well as Gene-Set Enrichment Analysis. This revealed that the T-cell receptor (TCR) and Notch pathways were the most enriched in mutations. In particular, variant T349P of NOTCH1, which confers a growth advantage to cells in which it is expressed, was detected in 12% of ALK+ and ALK- ALCL patient samples. Furthermore, we demonstrate that NPM-ALK promotes NOTCH1 expression through binding of STAT3 upstream of NOTCH1. Moreover, inhibition of NOTCH1 with γ-secretase inhibitors (GSIs) or silencing by shRNA leads to apoptosis; co-treatment in vitro with the ALK inhibitor Crizotinib led to additive/synergistic anti-tumour activity suggesting this may be an appropriate combination therapy for future use in the circumvention of ALK inhibitor resistance. Indeed, Crizotinib-resistant and sensitive ALCL were equally sensitive to GSIs. In conclusion, we show a variant in the extracellular domain of NOTCH1 that provides a growth advantage to cells and confirm the suitability of the Notch pathway as a second-line druggable target in ALK+ ALCL.

Recurrent gain-of-function mutations in the transcription factors STAT5A and much more in STAT5B were found in hematopoietic malignancies with the highest proportion in mature T- and natural killer-cell neoplasms (peripheral T-cell lymphoma). No targeted therapy exists for these heterogeneous and often aggressive diseases. Given the shortage of models for peripheral T-cell lymphoma, we mimicked graded STAT5A or STAT5B activity by expressing hyperactive Stat5a or STAT5B variants at low or high levels in the hematopoietic system of transgenic mice. Only high activity levels developed a lethal disease resembling human peripheral T-cell lymphoma. Neoplasia displayed massive expansion of CD8 ⁺ T-cells and destructive organ-infiltration. T-cells were cytokine-hypersensitive with activated memory CD8⁺ T-lymphocyte characteristics. Histopathology analysis and mRNA expression profiles revealed close correlation with distinct human peripheral T-cell lymphoma subtypes. Pronounced STAT5 expression and activity in patient samples of different subsets underlines the relevance of JAK/STAT as therapeutic target. JAK inhibitors or a selective STAT5 SH2 domain inhibitor induced cell death and Ruxolitinib blocked T-cell neoplasia in vivo. We conclude that enhanced STAT5A or STAT5B action both drive peripheral T-cell lymphoma development, defining both STAT5 molecules as targets for therapeutic intervention.

Anaplastic Large Cell Lymphoma (ALCL) is a T-cell malignancy predominantly driven by a hyperactive Anaplastic Lymphoma Kinase (ALK) fusion protein. ALK inhibitors such as crizotinib provide alternatives to standard chemotherapy with reduced toxicity and side effects. Children with lymphomas driven by NPM1-ALK fusion proteins achieved an objective response rate to ALK inhibition therapy of 54-90% in clinical trials. However, a subset of patients progress within the first 3 months of treatment. The mechanism for the development of ALK inhibitor resistance is unknown. Through genome-wide CRISPR activation and knockout screens in ALCL cell lines combined with RNA-seq data derived from ALK inhibitor relapsed patient tumors, we show that resistance to ALK inhibition by crizotinib in ALCL can be driven by aberrant upregulation of IL10RA. Elevated IL10RA expression rewires the STAT3 signaling pathway bypassing otherwise critical phosphorylation by NPM1-ALK. IL10RA expression does not correlate with response to standard chemotherapy in pediatric patients suggesting that combination of crizotinib with chemotherapy could prevent ALK-inhibitor resistance-specific relapse. Trials registered as NCT01979536/NCT02034981/UMIN000028075.

Genomic stability is crucial for cell life and transmitting genetic material is one of the primary tasks of the cell. The cell needs to be able to recognize any possible error and quickly repair it, and thus, cells have developed several mechanisms to detect DNA damage and promote repair during evolution. The DNA damage response (DDR) and DNA repair pathways ensure the control of possible errors that could impair the duplication of genetic information and introduce variants in the DNA. Endogenous and exogenous factors compromise genomic stability and cause dysregulation in the DDR and DNA repair pathways. Cancer cells often impair these mechanisms to overcome cellular barriers (cellular senescence and/or apoptosis), leading to malignancy. NPM (nucleophosmin)-ALK (anaplastic lymphoma kinase) is an oncogenic tyrosine kinase that is involved in the development of anaplastic large cell lymphoma (ALCL). NPM-ALK is known to be involved in the activation of proliferative and anti-apoptotic signaling pathways. New evidence reveals that NPM-ALK translocation also impairs the ability of cells to maintain the genomic stability through both DDR and DNA repair pathways. This review aims to highlight the role of the oncogenic tyrosine kinase NPM-ALK in the cell, and pointing to new possible therapeutic strategies.

BACKGROUND: Systemic Anaplastic Large Cell Lymphoma (sALCL) is comprised of two distinct T-cell non-Hodgkin lymphoma entities: ALK-positive (ALK+) ALCL and ALK-negative (ALK-) ALCL. These entities are characterized by either the presence of absence of an ALK-translocation. It has been reported that ALK+ ALCL has a better prognosis compared to ALK- ALCL, with a 5-year overall survival (OS) of 70-80% versus 15-45%, respectively. Furthermore, more than 25% of ALK+ ALCL patients undergo relapse. sALCL is a genetically heterogeneous disease whose genomic characterization has been improved through the implementation of high-throughput technologies. Despite this, the prognostic value of somatic mutations has been poorly described. Here we present the analysis of genetic aberrations of sALCL, shedding light on disease pathogenesis, novel diagnostic biomarkers and prognostic markers for the detection of refractory and/or relapsed patients which could hold clinical implications. METHODS: Formalin-fixed paraffin-embedded (FFPE) and/or fresh frozen tissue and related clinical information for 82 sALCL patients (47 ALK+ and 35 ALK-) were obtained after written informed consent from 5 centres across Europe. Using deep targeted DNA sequencing, the entire coding region of 275 selected genes (QIAseq Targeted DNA - Human Comprehensive Cancer Panel, Qiagen, Germany) was investigated in our retrospective cohort of patient' samples as well as 6 ALCL cell lines (Karpas-299, SU-DHL-1, DEL, SR786, FEPD and MAC2a). The average depth achieved across all the sequenced samples was approximately 2000x allowing the detection of aberrations with low allele frequencies (<10%) not usually detectable by other methods (e.g. Sanger sequencing or Whole Exome Sequencing). Moreover, we explored the clonal expansion in sALCL relapsed patients comparing the mutational status in matched samples at both diagnosis and relapse (n=4). RESULTS: ALK- patients in our cohort have a worse prognosis than ALK+, whereby the 7y-OS was 45.1% and 73.2%, respectively, although the 7 year progression free survival (PFS) was comparable at 57.1% for ALK+ and 42.4% for ALK- patients. As previously reported, ALK+ patients were significantly younger in our series than ALK- patients with an average age of 23.5 and 55.2 years, respectively. Among the 275 genes sequenced, 148 (53.8%) genes harbor at least one mutation throughout the entire patient and cell line cohort; almost 1/3 of mutated genes were shared between ALK- and ALK+ ALCL patients. On average 4.2 mutations per patient were detected in ALK- ALCL, a higher level than observed in ALK+ samples (2.7), most likely due to the oncogenic-driving role of the ALK-translocation in this group. Seventy-two out of 82 (88%) patients carried at least one single mutation within the genes analysed in our panel. The most recurrent genes mutated were TP53 in both sALCL categories (16% of sALCL patients), followed by LRP1B being prevalently mutated in ALK+ disease (15% of sALCL patients). As expected, STAT3 and JAK1 were mutated solely in the ALK- ALCL cohort and represent the most commonly mutated genes in this group. Prognostically, the most recurrent genes mutated in patients with a dismal outcome (dead and/or relapsed patients) were TP53, STAT3, EPHA5, JAK1, LRP1B, KMT2D, PRDM1 and SOCS1. Comparing samples at diagnosis versus relapse highlighted two key findings: firstly, the clonal expansion of TP53 mutated clones in relapsed ALCL patients and secondly, the acquired mutations in EPHA5 which were only detected at relapse. These data suggest their possible use as biomarkers associated with clonal evolution. CONCLUSION: This study provides information regarding the genetic landscape in sALCL across 275 select genes. We have confirmed the importance of known frequently mutated genes (STAT3 and JAK1) and describe novel recurrently mutated genes (LRP1B, EPHA5 and KMT2D). Mutations in STAT3 were more recurrent in ALK- ALCL patients with shorter OS, and clones harboring mutated TP53 and EPHA5 were detectable more often in relapsed sALCL thereby suggesting a possible driving role. Disclosures: Gambacorti-Passerini: Bristol-Meyers Squibb: Consultancy; Pfizer: Honoraria, Research Funding.