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Deregulation of JAK2 signaling underlies pcAECyTCL
included the evaluation of CNA in single patients by using array-based methods as well.6,7 Recurrent CNA uncovered by these studies include losses within 1p, 9p, 13q and 16p as well as gains within 7q, 8q and 17q, with loss of the region containing CDKN2A/B being the most frequent CNA.5 However, aside from the aforementioned chromo- somal imbalances, causative genetic changes in pcAECyTCL remain unknown.
Here, we present the first high-resolution genomic analy- sis of pcAECyTCL using whole-genome sequencing (WGS) and RNA sequencing (RNA-seq). We describe for the first time a number of genomic rearrangements, CNA and small- scale mutations with pathogenic relevance in this lym- phoma. In particular, our results suggest that overactivation of JAK2 signaling due to oncogenic changes in JAK2 and SH2B3, two genes with key roles in this signaling pathway, underlie predominantly pcAECyTCL. These findings have important implications for patient standard of care.
Methods
Patient selection and sequencing
Frozen tumor biopsies (≥70% tumor cells) from 12 patients with pcAECyTCL (Online Supplementary Figure S1; Online Supplementary Table S1) were subjected to WGS. Six samples of this cohort (i.e., AEC2-4/6/8/12) were additionally subjected to RNA-seq. Sequencing, data processing and DNA/RNA analyses are described in the Online Supplementary Appendix (Online Supplementary Figures S2 and S3; Online Supplementary Tables S2 to S9). Diagnosis was performed by an expert panel of dermatolo- gists/pathologists in accordance with the WHO-EORTC classifi- cation for primary cutaneous lymphomas.1,2 Patient material was approved by the Institutional Review Boards of the Fondazione IRCCS Ca' Granda Ospedale Maggiore Policlinico and Leiden University Medical Center. Informed consent was obtained from patients in accordance with the declaration of Helsinki.
Validation of structural genomic alterations and small-scale mutations
Select rearrangements, interstitial deletions and single nucleotide variants (SNV) were validated by Sanger sequencing, droplet digital polymerase chain reaction (ddPCR) and/or fluores- cence in situ hybridization (FISH). Details of the validation exper- iments are included in the Online Supplementary Appendix (Online Supplementary Figures S4 to S9; Online Supplementary Table S10).
Immunohistochemistry
Formalin-fixed paraffin-embedded (FFPE) tissue sections were immunohistochemically stained with primary antibodies against phospho-STAT3 (Cell Signaling Technology, Cat.No. 9145) or phospho-STAT5 (Cell Signaling Technology, Cat.No. 9359) using Dako REAL detect system (Dako, Cat.No. K5005), counter- stained in Mayer’s hematoxylin solution and coverslipped using Vectamount (Vector Laboratories, Cat.No. H5000).
Cell culture, fusion gene construction and viral transduction
Ba/F3 cells (DSMZ, Cat.No. ACC-300) were used for function- al experiments. Parental Ba/F3 cells were cultured in RPMI-1640 (10% heat-inactivated fetal bovine serum, 10 ng/mL interleukin- 3 [IL3]) at 37° C with 5% CO2 in a humidified atmosphere. JAK2 fusions (i.e., TFG-JAK2, PCM1-JAK2, KHDRBS1-JAK2) and con- trol genes (i.e., eGFP, TFG-MET) were constructed and inserted into a lentiviral vector using the method described by Lu et al.8
Primers, templates and vectors used for fusion gene construction are detailed in the Online Supplementary Appendix (Online Supplementary Tables S11 to S13). Lentiviral particles were pro- duced in HEK-293T cells, quantified by p24 enzyme-linked immunosorbent assay (ELISA), and transduced into Ba/F3 cells at MOI-9 with lipofectamine. Successfully transduced Ba/F3 cells were selected with puromycin (2.5 mg/mL) for 3 days.
Ba/F3 cell viability and inhibitor assays
Cell viability of parental and transduced Ba/F3 cells was deter- mined 7 days after IL3 withdrawal by MTT assay (Promega, Cat.No. G4000). Inhibitor assays were performed by treating IL3-independent Ba/F3 cells expressing fusion genes with ruxoli- tinib or AZD1480 at seven different concentrations for 72 hours and measuring cell viability by MTT assay.
Western blots
The effect of JAK1/2 inhibitors ruxolitinib and AZD1480 on JAK2 and STAT5 phosphorylation was evaluated by western blotting. Cells were washed to remove traces of serum and incu- bated with inhibitor for 90 minutes. Cells were lysed in SDS lysis buffer containing protease inhibitors and separated by SDS- PAGE. Antibodies employed were anti-JAK2 (Abcam, Cat.No. ab108596), anti-phospho-JAK2 (Cell Signaling Technology, Cat.No. 3776), anti-STAT5 (Cell Signaling Technology, Cat.No. 94205), anti-phospho-STAT5 (Cell Signaling Technology, Cat.No. 9351) and anti-GAPDH (Cell Signaling Technology, Cat.No. 2118).
Results
JAK2 fusions are prominent in a complex landscape of rearrangements
The analysis revealed a heterogeneous and complex landscape of genomic rearrangements (total events, 426; range, 10-65; mean/patient ± standard deviation [SD], 36±21) (Figure 1; Figure 2A; Online Supplementary Figure S3). Fifty-three percent of events were interchromosomal (range/patient, 27-80%) (Figure 2B). The majority of rearrangements (77%) disrupted either one or two anno- tated genes, while the rest (23%) disrupted nongenic regions (Figure 2C). Four patients, AEC6, AEC7, AEC10 and AEC12, displayed complex rearrangements (chro- mothripsis/chromoplexy-like) affecting chromosomes 13, 10, 1/9/12 and 4, respectively (Figure 2D; Online Supplementary Figure S3 and S10). We observed a total of 305 rearranged genes, 59 of which are implicated in neo- plasms at present (Online Supplementary Table S14). Gene ontology analysis revealed that rearranged genes encode principally (ngenes=91 of 305) proteins with roles in signal transduction (i.e., hydrolases, transferases, enzyme modu- lators, receptors) and transcriptional regulation (i.e., tran- scription factors, chromatin regulators) (Figure 2E; Online Supplementary Tables S15 and S16). Out of seventeen recur- rently rearranged genes detected in our cohort (npatients =2 or 3) (Online Supplementary Table S17), six are established can- cer genes with important functions in the regulation of the cell cycle (i.e., MYC, RB1), chromatin remodeling (i.e., BAZ1A) and the JAK-STAT pathway (i.e., JAK2, PTPRC, SH2B3) (Figure 1; Online Supplementary Figures S4 and S5).
The JAK-STAT pathway, a frequent driver of hemato- logical neoplasms, was the only cytokine-elicited signal transduction pathway impacted by rearrangements in pcAECyTCL. Fusion genes involving JAK2 were detected
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