Page 102 - 2019_04-Haematologica-web

M.R. Sapienza et al.
Introduction
Whole-exome sequencing analysis
We performed paired-end sequencing of matched tumor/normal DNA samples (9 cases), tumor only DNA samples (5 cases), and the CAL-1 cell line (Online Supplementary Table S3) using the TruSeq Exome Kit and Nextera Rapid Capture Exome kit (Illumina). Further details are available in the Online Supplementary Appendix.
Sanger sequencing
We used Sanger sequencing to analyze two candidate nonsense somatic mutations of SUZ12 and ASXL1 occurring in 2 patients, respectively, as described in the Online Supplementary Appendix.
Targeted sequencing
We performed MiSeq targeted sequencing (Illumina) of the 14 BPDCN tumor patients, 7 normal matched saliva samples and the CAL-1 cell line, already analyzed by WES. More bioinformatics details are provided in the Online Supplementary Appendix and Online Supplementary Tables S4 and S5.
RNA sequencing analysis
Five BPDCN cases studied by WES and targeted sequenc- ing had sufficient material for RNA extraction and sequenc- ing; these samples represented the RNA sequencing (RNA- seq) discovery set. We also collected an additional 4 BPDCN cryopreserved cutaneous biopsies, sufficient only for RNA sequencing experiments, used as an RNA-seq extension set. RNA of 4 normal plasmacytoid dendritic cell (pDCs) sam- ples was purchased from AllCells (Alameda, CA, US) and used for comparison. For details, see Online Supplementary Table S6 and the Online Supplementary Appendix.
Pathology tissue-chromatin immunoprecipitation sequencing
The BPDCN_25 and BPDCN_37 patients were provided with one skin biopsy: half was cryopreserved and used for WES, targeted and RNA sequencing analysis, and the other half was fixed in formalin, included in paraffin and used for pathology tissue-chromatin immunoprecipitation (PAT- ChIP) sequencing analysis. PAT-ChIP experiments were per- formed as in Fanelli et al.26 Further details are available in the Online Supplementary Appendix.
CAL-1 cell line
CAL-1, a BPDCN cell line27 was cultured as reported pre- viously.18 The CAL-1 gene expression profile of a previous study was used17 (http://www.ncbi.nlm.nih.gov/geo/query/ acc.cgi?acc=GSE62014).
Mouse model and in vivo treatments
Experiments were carried out on 6-8-week old non-
obese diabetic severe combined immunodeficient NOD/SCID interleukin-2 receptor g (IL-2Rg)–null (NSG) mice, as previously reported.13 All animal experiments were carried out in accordance with the Italian laws in force (Legislative Decree 26/14 and subsequent amend- ments) and institutional guidelines. All in vivo studies were ratified by the Italian Ministry of Health. For induction of BPDCN in mice, 5000 CAL-1 cells were injected intra- venously (i.v.) through the lateral tail vein in non-irradiat- ed mice. Engrafted mice were then treated with borte- zomib, 5’-azacytidine, decitabine and romidepsin, as detailed in the Online Supplementary Appendix.
Blastic plasmacytoid dendritic cell neoplasm (BPDCN) is a rare malignancy derived from precursors of plasma- cytoid dendritic cells.1-4 It has no clear racial or ethnic predisposition and more often affects elderly males (male/female ratio 3.3:1; mean/median age at diagnosis: 61-67 years). BPDCN patients usually respond to first- line chemotherapy, but despite this they almost invari- ably relapse and display a dismal prognosis with a medi- an overall survival (OS) ranging from 10 to 19 months.2 No standardized therapeutic approach has so far been established for BPDCN, even if hematopoietic stem cell transplantation has been shown to achieve remission in selected patients.5-6 Therefore, the development of effec- tive treatments still represents an unmet need.7 The pathobiology of BPDCN is poorly understood and the number of reports exploring its molecular features is still limited.8-21 Recent advances in the understanding of the BPDCN molecular landscape have paved the way for novel treatment approaches based on the inhibition of the BCL2 protein,22 the activation of the cholesterol efflux,23 the repression of the Bromodomain-containing protein 4 (BRD4),24 and binding to the interleukin-3 receptor (IL3R).25 All these potential therapeutic options (which are worthy of further evaluation) have mainly emerged from the analysis of the BPDCN transcriptome or from its antigenic repertoire. The genomic landscape of BPDCN has not been well investigated, and only a few studies have explored the mutational events occur- ring in BPDCN, mainly through targeted sequencing approaches.14,16,19,20
Unfortunately, these have not offered any novel prospects of treatment options.
In this study, we performed whole-exome sequencing (WES) of 14 BPDCN samples and of the BPDCN-derived CAL-1 cell line to look for specific BPDCN genetic vulner- abilities that may support the design of new therapeutic strategies. The WES mutational findings were comple- mented by copy number variant (CNV) analysis, RNA and pathology tissue-chromatin immunoprecipitation (PAT- ChIP) sequencing results. The integration of data allowed us to identify a successful combinatorial therapy based on epigenetic drugs able to control disease progression in a rapidly progressive BPDCN xenograft model.
Methods
Blastic plasmacytoid dendritic cell neoplasm samples
We collected 14 BPDCN cryopreserved cutaneous biop- sies at diagnosis, 9 matched saliva samples and the BPDCN patient-derived cell line, CAL-1. The pathological cases were evaluated as previously described17 and diag- nosed by experienced hematopathologists (CA, EB, FF, LC, MP, ES, CT, MT, and SAP) according to World Health Organization diagnostic criteria.2 Informed consent was obtained from each patient in accordance with the Ethical Review Board of the Department of Experimental, Diagnostic, and Specialty Medicine of the University of Bologna, Italy, and the Declaration of Helsinki. DNA was extracted as reported in the Online Supplementary Appendix. The main clinical, immunohistochemical and cytogenetic features of the BPDCN patients are shown in Online Supplementary Tables S1 and S2.
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