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NOTCH1 and the pathobiology of ALK-positive ALCL
using the histoscore system. Stained slides were scored qualita- tively for the intensity of staining and classified as showing nega- tive, weak, moderate or strong staining (to qualify for ‘moderate’ or ‘strong’ staining, at least 10% of cells had to stain positive). Analysis of event-free survival was performed as described previ- ously,19 grouping negative and weak staining into ‘low cleaved NOTCH1 expression’ and moderate and strong into ‘high cleaved NOTCH1 expression’.
Statistical analyses
All experiments were executed in biological triplicates. The MTT, RealTimeGlo, apoptosis, cell cycle and quantitative poly- merase chain reaction assays were additionally executed with technical triplicates. All plots are representative of the mean of the biological replicates, while the error bars represent the standard deviation. Two-tailed t-tests were used to calculate the P-value when comparing two samples (multiple comparisons were cor- rected using the Holm-Sidak method); when comparing more than two samples, two-way analysis of variance (ANOVA) was used (again, multiple comparisons were corrected using the Holm- Sidak method). Statistical tests were conducted using GraphPad PRISM 8 (Graphpad).
Results
The genomic profile of anaplastic large cell lymphoma
Eighteen ALK+ ALCL exomes sequenced in this study in addition to seven previously sequenced samples9 (Online Supplementary Table S1) were analyzed. The samples com- prised 17 pediatric cases (≤18 years) and eight adult cases (Online Supplementary Table S4); a flowchart illustrating the cohorts of patients is shown in Online Supplementary Figure S1. All patient samples were collected at diagnosis. Data regarding variants found in at least a quarter of the patients are summarized in Figure 1, which shows that the most commonly mutated genes in both adult and pediatric cases were TYW1B, DEFB132 and KCNJ18 (the full list of vari- ants can be found in Online Supplementary Table S6). None of the variants in these genes has been reported previously in hematologic malignancies and were not studied further here. Two patients presented with one mutation each in TP53 (COSMIC ID: COSM3958801 and COSM9969). We also studied copy number variations, but found no novel events larger than 100,000 bp present consistently in more than one sample at a sequencing depth of at least 50x (Online Supplementary Figure S2A, Online Supplementary Table S7), as previously observed.4 Among previously reported alterations in ALCL, a single copy gain on chro- mosome 7 was observed in three patient tumor samples (S3, S9 and S15)5 and a single copy loss on chromosome 17p was also seen in three patients (S9, S14 and S57).6
The most predominant single nucleotide variants
are non-synonymous and are present at higher levels in patients who subsequently relapsed
The majority of somatic variants detected in the 25 tumor samples were non-synonymous single nucleotide variants (39.4%), in keeping with a previous publication reporting that the ALK+ ALCL genome is largely stable.4 Single nucleotide variants were followed in frequency by frameshift and non-frameshift deletions and splice variants (24.1%, 10.8% and 10.6%, respectively), while the germline genome of ALCL patients points to an over- whelming presence of single nucleotide polymorphisms
(89.3%) (Online Supplementary Figure S2B). The proportion of each type of variant detected differed between patient tumors (Figure 2B), although in general, pediatric patients known to have relapsed (n=9) had a significantly higher proportion of non-synonymous single nucleotide variants than patients who did not (n=9; P<0.0001) (Figure 2B), sug- gesting that a high percentage of non-synonymous single nucleotide variants at diagnosis may be indicative of relapse, although this requires validation in a larger dataset of patients treated with comparable treatment protocols.
Deficiency of DNA repair mechanisms and spontaneous deamination of 5-methyl cytosine are identified as signatures of anaplastic large cell lymphoma
Online Supplementary Figure S2C shows the prevalence, in representative patient S57, of the 96 variant types that were used to derive the mutational signatures (Online Supplementary Figure S2D). Examining the type of muta- tions in the patients for whom matched peripheral blood was available (n=11), showed an enrichment for muta- tional signatures 1, 3, 12 and 2620 (Figure 2C). Interestingly, 1A is a signature based on the prevalence of C>T transi- tions at NpCpG trinucleotides and is associated with spontaneous deamination of 5-methyl-cytosine,21 whereas signature 3 has its roots in homologous recombination deficiency during DNA double-strand break repair.20 The etiology of signature 12 has not yet been identified, although signature 26 is associated with a breakdown in DNA mismatch repair. The combination of signatures 3 and 26 may indicate, from an evolutionary perspective, how ALCL tumors accumulate mutations. Comparable patterns were found when comparing signatures to the COSMIC signature database22 (data not shown). There was no detectable difference between the mutational signature of pediatric (n=4) or adult (n=7) ALK+ ALCL patients (data not shown).
Gene set enrichment analysis confirms the importance of T-cell receptor signaling, but also of the Notch pathway in ALK+ anaplastic large cell lymphoma pathobiology
Gene set enrichment analysis (GSEA) of mutated genes showed that TCR signaling and Notch pathways are enriched across all five databases used (Figure 2D; Online Supplementary Table S8). Further analysis of the domains frequently found in the mutated genes revealed an enrich- ment in proteins with epidermal growth factor (EGF)-like or calcium-ion binding domains (Figure 2E), two features of the NOTCH1 protein, and indeed the locus of both of the NOTCH1 mutations identified in this study (see below). Twenty of the 25 patient tumors carry mutations in proteins of the Notch pathway with a range of one to four and a median of two mutations per patient (Online Supplementary Table S8). Furthermore, reactome network clustering analysis23 showed TP53 as a key node, which is not unexpected as TP53 has been reported to play a key role in the pathogenesis of ALCL24 (Online Supplementary Figure S2E). Given the importance of the Notch pathway in T-cell biology, particularly in the developing thymus, which we proposed tp be the origin of ALK+ ALCL,25 and the previous implication of the NOTCH1 pathway in the pathogenesis of ALCL,8 the NOTCH1 mutations detected and the NOTCH1 pathway were explored for their role in the pathogenesis of ALK+ ALCL.
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