Original Article Clinical Genomic Profiling Identifies TYK2 Mutation and Overexpression in Patients With Neurofibromatosis Type 1-Associated Malignant Peripheral Nerve Sheath Tumors Angela C. Hirbe, MD, PhD1; Madhurima Kaushal, MS2; Mukesh Kumar Sharma, PhD2; Sonika Dahiya, MD2; Melike Pekmezci, MD3; Arie Perry, MD3,4; and David H. Gutmann, MD, PhD5 BACKGROUND: Malignant peripheral nerve sheath tumors (MPNSTs) are aggressive sarcomas that arise at an estimated frequency of 8% to 13% in individuals with neurofibromatosis type 1 (NF1). Compared with their sporadic counterparts, NF1-associated MPNSTs (NF1-MPNSTs) develop in young adults, frequently recur (approximately 50% of cases), and carry a dismal prognosis. As such, most individuals affected with NF1-MPNSTs die within 5 years of diagnosis, despite surgical resection combined with radiotherapy and che- motherapy. METHODS: Clinical genomic profiling was performed using 1000 ng of DNA from 7 cases of NF1-MPNST, and bioinformatic analyses were conducted to identify genes with actionable mutations. RESULTS: A total of 3 women and 4 men with NF1-MPNST were identified (median age, 38 years). Nonsynonymous mutations were discovered in 4 genes (neurofibromatosis type 1 [NF1], ROS proto-oncogene 1 [ROS1], tumor protein p53 [TP53], and tyrosine kinase 2 [TYK2]), which in addition were mutated in other MPNST cases in this sample set. Consistent with their occurrence in individuals with NF1, all tumors had at least 1 mutation in the NF1 gene. Whereas TP53 gene mutations are frequently observed in other cancers, ROS1 mutations are common in melanoma (15%-35%), anoth- er neural crest-derived malignancy. In contrast, TYK2 mutations are uncommon in other malignancies (<7%). In the current series, re- current TYK2 mutations were identified in 2 cases of NF1-MPNST (30% of cases), whereas TYK2 protein overexpression was observed in 60% of MPNST cases using an independently generated tissue microarray, regardless of NF1 status. CONCLUSIONS: Clinical geno- mic analysis of the current series of NF1-MPNST cases found that TYK2 is a new gene mutated in MPNST. Future work will focus on examining the utility of TYK2 expression as a biomarker and therapeutic target for these cancers. Cancer 2016;000:000–000. VC 2016 American Cancer Society. KEYWORDS: cancer predisposition, neurofibromatosis, ROS proto-oncogene 1 (ROS1), sarcoma, tyrosine kinase 2 (TYK2). INTRODUCTION Neurofibromatosis type 1 (NF1) is one of the most common inherited tumor predisposition syndromes, affecting 1 in 2500 individuals worldwide.1 As such, individuals with NF1 begin life with 1 mutated (germline) copy and 1 functional copy of the NF1 gene in every cell in their body.2 The presence of this germline mutation significantly increases their risk of developing cancer because tumorigenesis requires only somatic loss of the remaining functional NF1 gene. In this re- gard, individuals with NF1 are prone to the development of central and peripheral nervous system malignancies, including malignant peripheral nerve sheath tumors (MPNSTs). MPNSTs are aggressive soft tissue sarcomas3 that arise in individuals with NF1 at an estimated frequency of 8% to 13%. In contrast to sporadic MPNSTs, which arise in adults during their 60s and 70s, NF1-associated MPNSTs (NF1- MPNSTs) tend to occur in young patients, usually in their 20s and 30s.4 These cancers are composed of neoplastic Schwann cells, and in the setting of NF1, most often arise from a benign precursor lesion, termed a plexiform neurofibro- ma. When MPNSTs are diagnosed, they initially are treated with surgery and radiotherapy, and in some situations, che- motherapy is added. However, despite aggressive initial management with multimodality therapy, approximately 50% of affected individuals will experience cancer recurrence or metastatic disease, and the majority of patients die within 5 years Corresponding author: David H. Gutmann, MD, PhD, Department of Neurology, Washington University, Box 8111, 660 S. Euclid Ave, St. Louis, MO 63110; Fax: (314) 362-2388; [email protected] 1Division of Medical Oncology, Department of Medicine, Washington University School of Medicine, St. Louis, Missouri; 2Department of Pathology and Immunolo- gy, Washington University School of Medicine, St. Louis, Missouri; 3Department of Pathology, University of California at San Francisco School of Medicine, San Francisco, California; 4Department of Neurological Surgery, University of California at San Francisco School of Medicine, San Francisco, California; 5Department of Neurology, Washington University, St. Louis, Missouri Additional supporting information may be found in the online version of this article. DOI: 10.1002/cncr.30455, Received: June 9, 2016; Revised: August 22, 2016; Accepted: October 24, 2016, Published online Month 00, 2016 in Wiley Online Library (wileyonlinelibrary.com) Cancer Month 00, 2016 1 Original Article of their diagnosis.5-7 Moreover, metastatic tumors are par- eosin ethanol-based staining protocol. Within 10 minutes of ticularly difficult to treat because they inevitably become air-drying,theslidewasplacedontheLCMstageoftheArc- resistant to chemotherapy. Given the poor prognosis of turus PixCell instrument (Thermo Fisher Scientific, Wal- these tumors, it is essential that better strategies be devel- tham, Mass) for microdissection. The desired cells were oped to detect and treat these aggressive malignancies. microdissected into the cap of a 500-lL, safe-lock tube filled To identify improved treatments for patients with with 50 lL of ALT buffer (Qiagen, Valencia, Calif). This NF1-MPNSTs, several groups have used genetically engi- procedure was repeated on the next slide until a total of 2000 neered mouse models of these cancers, resulting in the dis- to 5000 cells of interest had been captured for each case. covery of new biologically targeted therapies.8-14 Based on these basic discovery efforts, mechanistic target of rapa- DNA Extraction mycin (mTOR), mitogen-activated kinase kinase (MEK), LCM-derived DNA samples were isolated from 2000 to and heat shock protein 90 (HSP90) inhibitors have all en- 5000 cells using a QIAamp DNA Micro Kit (Qiagen cate- tered the clinical trials pipeline as promising targeted thera- gory 56304; Qiagen) after RNase A treatment, according pies for NF1 tumors (ClinicalTrials.gov identifiers to the manufacturer’s instructions. DNA was quantified NCT01661283, NCT01885195, and NCT020088770). using a NanoDrop 2000 spectrophotometer (Thermo As a complementary approach to mouse model-driven dis- Fisher Scientific). covery,15-17 in the current study, we leveraged clinical-grade genomic profiling on MPNSTs from 7 subjects with NF1 Sequencing to identify new potentially actionable gene mutations. For these studies, we employed next-generation sequenc- ing using a protocol similar to Clinical Laboratory Im- MATERIALS AND METHODS provement Amendments-approved mutation testing Patients available for patients with cancer treated at the study insti- Tumors were obtained from individuals diagnosed with tution (Genomics and Pathology Services; https://gps. NF1 according to established criteria18 and treated at the wustl.edu/patient care/). The list of sequenced genes in Washington University/St. Louis Children’s Hospital this set are listed in Supporting Information Table 1. In Neurofibromatosis Clinical Program. This study was per- brief, genomic DNA was sonicated to an average size of formed under active Human Studies Protocols approved approximately 200 base pairs. The fragments were blunt by the Institutional Review Boards at each respective insti- ended, “A” tailed, and ligated to Illumina sequencing tution in accordance with the 1964 Declaration of adapters (Illumina Inc, San Diego, Calif). The ligated Helsinki and its later amendments or comparable ethical fragments were amplified for 7 polymerase chain reaction standards. Tumor DNA samples were obtained from cycles. Exome targets were enriched with Agilent Custom formalin-fixed paraffin-embedded (FFPE) blocks or fro- SureSelect reagents (Agilent Technologies, Santa Clara, zen tissue (when available) obtained at the time of surgical Calif) according to the manufacturer’s protocols. resection or biopsy. Enriched fragments were amplified for 14 cycles with pri- mers that incorporate a unique indexing sequence tag. Laser Capture Microdissection The resulting library fragments were sequenced from each Images of hematoxylin and eosin-stained slides were creat- end (paired-end reads) for 101 bases using an Illumina ed on an Aperio ScanScope XT (Leica Biosystems, Buffalo HiSeq-2500 instrument (Illumina Inc). Grove, Ill). The Aurora mScope viewer (Aurora Interac- tive Ltd, Montreal, Quebec, Canada) was used to view the Analysis images and obtain images at 320 magnification. A pa- Sequence reads were mapped to the human reference ge- thologist examined a digitized hematoxylin and eosin- nome (GRCh37/hg19) using the Novoalign tool (http:// stained slide to determine and outline tumor nests, direct- www.novocraft.com/products/novoalign/).19 Marking and ing the laser capture microdissection (LCM) to areas of removing polymerase chain reaction duplicates were per- high tumor cell content on the adjacent FFPE sections. A formed using the PICARD tool (http://broadinstitute. total of 5 to 10 sections (10-lm thick) from each FFPE github.io/picard/). Variants were determined using the block were cut on a microtome (Leica RM 2255; Leica GATK UnifiedGenotyper (https://www.broadinstitute. Biosystems), air-dried for 2 hours,