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H. Larose et al.
therapy for future use in the circumvention of ALK inhibitor resistance. Indeed, crizotinib-resistant and -sen- sitive ALCL were equally sensitive to γ-secretase inhibitors. In conclusion, we show a variant in the extracel- lular 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.
Introduction
Systemic anaplastic large cell lymphoma (ALCL) is a T- cell malignancy accounting for approximately 15% of all cases of pediatric lymphoma and 1-2% of adult lym- phomas.1 The majority of pediatric cases (>90%) express NPM-ALK, the result of the t(2;5)(p23;q35) translocation, creating a fusion between the nucleolar phosphoprotein gene nucleophosmin1 (NPM1) and anaplastic lymphoma kinase (ALK), leading to the ALK+ ALCL diagnostic entity, although other ALK fusion proteins also exist.2,3 ALK fusion proteins induce the activation of several downstream sig- naling pathways involved in oncogenesis, including PI3K, ERK1/2 MAP kinase, and JAK-STAT.
Genetic studies conducted to date have not revealed con- sistent genetic abnormalities among ALK+ ALCL, although a higher frequency of genomic gains and losses has been associated with a poorer prognosis.4-6 Genomic gains include the region encoding the NOTCH1 locus,4 which may explain why NOTCH1 is expressed in the majority of ALK+ ALCL and in some ALK– ALCL.7,8 In contrast, for ALK– ALCL, mutations in the JAK1/STAT3 pathway have been described.9
ALK+ ALCL is largely a chemosensitive malignancy, although despite good initial responses, the relapse rate reaches 50% independently of the chemotherapy regimen used;10-14 therefore, new therapies are needed for patients who do not respond to standard chemotherapy. There is also a need for less toxic treatment schedules for low-risk patients. Crizotinib, a small molecule ATP-competitive inhibitor of ALK/MET/ROS1 which is currently in clinical trials (NCT01606878, NCT02034981) for pediatric ALK+ lymphoid malignancies, given as monotherapy produces remission in more than 80% of relapsed patients, although rapid relapse on discontinuation of therapy has been report- ed.11,15-17 As such, second-line treatment and combination therapies for relapsed patients are still required. We there- fore conducted whole exome sequencing (WES) of 25 ALK+ ALCL tumors, validating data in a further 78 cases of ALCL to understand disease pathology and to uncover novel tar- gets for therapy. Beside the T-cell receptor (TCR) pathway, the most commonly affected signaling axis is that involving NOTCH1, of which a T349P variant provides a growth advantage to cells. Furthermore, NOTCH1 is expressed as a consequence of NPM-ALK-driven STAT3 activity, a key sig- naling node in ALCL. Accordingly, the NOTCH1 pathway provides a target for second-line therapy, whereby γ-secre- tase inhibitors (GSI) show synergistic activity with inhibitors of ALK, and are efficacious as single agents in ALK-inhibitor resistant cell lines. Finally, we show evidence that NOTCH1 is a biomarker predictive of relapse risk.
Methods
Patients’ samples
Patients’ tumor tissues (at initial presentation) and matched peripheral blood DNA were obtained following patient/parental
consent according to both the Declaration of Helsinki and local guidelines from the following institutions: Children’s Cancer and Leukaemia Group tissue bank, Newcastle, UK (Online Supplementary Table S1); Institut Universitaire du Cancer Toulouse, France; University Hospital Brno, Czech Republic; Biobank of the Medical University of Graz, Austria; Belarusian Center for Pediatric Oncology, Hematology and Immunology, Minsk, Belarus; Justus-Liebig University, Giessen, Germany; and Our Lady’s Children’s Hospital, Crumlin, Ireland (Online Supplementary Table S2). All tissues were obtained and processed with full ethical approval (NHS Research ethics committee reference numbers 07/Q0104/16, 06/MRE04/90 and 08/H0405/22+5).
The patient tissue microarray used here has been described else- where.18 Briefly, formalin-fixed paraffin-embedded (FFPE) tissue specimens from pediatric, patients with NPM-ALK+ ALCL treated in the Berlin-Frankfurt-Munster (BFM) group study NHL-BFM90, NHL-BFM95 or patients enrolled in the European intergroup trial ALCL99 between August 1998 and December 2008 were obtained from both male and female children (Online Supplementary Table S3) with informed consent and in accordance with the Declaration of Helsinki. Eligibility was confirmed by demonstration of NPM- ALK positivity of the tumor either by NPM-ALK polymerase chain reaction, two-color fluorescence in situ hybridization for the t(2;5), or nuclear and cytoplasmic staining for ALK. The inclusion criteria were fulfilled by 89 patients.
Whole exome sequencing
DNA was extracted from fresh-frozen tissue from patients (n=18; with a tumor content >90%) and matched peripheral blood from four patients using the QIAgen DNAeasy Blood and Tissue Kit (Qiagen, Hilden, Germany), following the manufacturer’s instructions. Library preparation was conducted using the Nextera Rapid Capture Exome Kit before samples were sequenced, at either the Washington State University Core (Illumina HiSeq2500) or the Molecular Diagnostics and Personalized Therapeutics Unit, University of Ha’il (Illumina MiSeq) (Online Supplementary Table S4). Sequencing data are available at the Sequence Read Archive (https://www.ncbi.nlm.nih.gov/sra) under accession number PRJNA491296. The bioinformatic processing is detailed in the Online Supplementary Methods.
Immunohistochemistry, pathology and quantification
Immunohistochemistry was performed on FFPE sections with the conventional avidin–biotin–peroxidase method. Heat antigen retrieval was performed using citrate buffer, pH 6.1. Endogenous peroxidases were quenched by incubating sections in 3% H O in
22 phosphate-buffered saline (PBS) for 10 min. An avidin/biotin and
a protein block were subsequently performed. Primary antibodies (Online Supplementary Table S5) were added in 1% bovine serum albumin/PBS at 4°C overnight. Slides were incubated with biotin- conjugated secondary antibodies and then with horse radish per- oxidase (HRP) using the IDtect Super Stain System – HRP and developed under visual control using aminoethyl carbazole. Hemalaun counterstaining was performed and slides mounted with AquaTex. Sections were washed with PBS three times in between each step. Stained slides were assessed for cleaved NOTCH1 staining by an experienced pathologist (blinded with respect to clinicopathological parameters and patients’ outcome)
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