NRG1 Gene Fusions Are Recurrent, Clinically Actionable Gene Rearrangements in KRAS Wild-Type Pancreatic Ductal Adenocarcinoma Martin R

Published OnlineFirst May 8, 2019; DOI: 10.1158/1078-0432.CCR-19-0191

Precision Medicine and Imaging Clinical Cancer Research NRG1 Gene Fusions Are Recurrent, Clinically Actionable Gene Rearrangements in KRAS Wild-Type Pancreatic Ductal Adenocarcinoma Martin R. Jones1, Laura M. Williamson1, James T. Topham2, Michael K.C. Lee3, Angela Goytain4, Julie Ho4, Robert E. Denroche5, GunHo Jang5, Erin Pleasance1, Yaoquing Shen1, Joanna M. Karasinska2, John P. McGhie3, Sharlene Gill3, Howard J. Lim3, Malcolm J. Moore6, Hui-li Wong3, Tony Ng4, Stephen Yip4, Wei Zhang1, Sara Sadeghi1, Carolyn Reisle1, Andrew J. Mungall1, Karen L. Mungall1, Richard A. Moore1, Yussanne Ma1, Jennifer J. Knox5,6, Steven Gallinger5,7, Janessa Laskin3, Marco A. Marra1,8, David F. Schaeffer2,4, Steven J.M. Jones1,8,9, and Daniel J. Renouf2,3

Abstract

Purpose: Gene fusions involving neuregulin 1 (NRG1) have Results: Three of 47 (6%) patients with advanced pancreatic been noted in multiple cancer types and have potential ther- ductal adenocarcinoma were identified as KRAS wild type by apeutic implications. Although varying results have been whole-genome sequencing. All KRAS wild-type tumors were reported in other cancer types, the efficacy of the HER-family positive for gene fusions involving the ERBB3 ligand NRG1. kinase inhibitor afatinib in the treatment of NRG1 fusion– Two of 3 patients with NRG1 fusion–positive tumors were positive pancreatic ductal adenocarcinoma is not fully treated with afatinib and demonstrated a significant and rapid understood. response while on therapy. Experimental Design: Forty-seven patients with pancreatic Conclusions: This work adds to a growing body of evidence ductal adenocarcinoma received comprehensive whole- that NRG1 gene fusions are recurrent, therapeutically action- genome and transcriptome sequencing and analysis. Two able genomic events in pancreatic cancers. Based on the patients with gene fusions involving NRG1 received afatinib clinical outcomes described here, patients with KRAS wild- treatment, with response measured by pretreatment and post- type tumors harboring NRG1 gene fusions may benefit from treatment PET/CT imaging. treatment with afatinib.

1BC Cancer, Canada's Michael Smith Genome Sciences Centre, Vancouver, British Columbia, Canada. 2Pancreas Centre British Columbia, Vancouver, Introduction 3 Canada. BC Cancer, Division of Medical Oncology, Vancouver, British Columbia, Oncogenic gene fusions involving neuregulin 1 (NRG1) are Canada. 4Department of Pathology and Laboratory Medicine, Vancouver Gen- recurrent across a variety of different cancer types and have eral Hospital, Vancouver, British Columbia, Canada. 5PanCuRx Translational Research Initiative, Ontario Institute for Cancer Research, Toronto, Ontario, emerged as clinically actionable genomic events in lung cancers Canada. 6Division of Medical Oncology, Princess Margaret Cancer Centre, and cholangiocarcinoma (1–4). As part of the ongoing Person- Toronto, Ontario, Canada. 7Lunenfeld Tanenbaum Research Institute, Mount alized OncoGenomics (POG; NCT02155621) and Prospectively Sinai Hospital, Toronto, Ontario, Canada. 8Department of Medical Genetics, Defining Metastatic Pancreatic Ductal Adenocarcinoma Subtypes 9 University of British Columbia, Vancouver, British Columbia, Canada. Depart- by Comprehensive Genomic Analysis (PanGen; NCT02869802) ment of Molecular Biology and Biochemistry, Simon Fraser University, Vancou- clinical trials at our site, patients with advanced cancers undergo ver, British Columbia, Canada. comprehensive genomic analysis including whole-genome Note: Supplementary data for this article are available at Clinical Cancer sequencing and transcriptome analysis (WGTA). We have previ- Research Online (http://clincancerres.aacrjournals.org/). ously described a patient with cholangiocarcinoma that harbored Clinical trial information: Personalized OncoGenomics (POG) Program of an ATP1B1–NRG1 gene fusion who responded favorably to British Columbia: Utilization of Genomic Analysis to Better Understand Tumour fi treatment with the HER-family kinase inhibitor afatinib (2). Heterogeneity and Evolution (NCT02155621); Prospectively De ning Metastatic NRG1 Pancreatic Ductal Adenocarcinoma Subtypes by Comprehensive Genomic However, not all tumors with fusions are responsive to Analysis (PanGen; NCT02869802). afatinib, as evidenced by a lack of therapeutic benefit reported in a series of NRG1 fusion–positive lung cancers (4), highlighting the J. Laskin, M.A. Marra, D.F. Schaeffer, S.J.M. Jones, and D.J. Renouf co-supervised this work. need for further research in this area. At a genomic level, pancreatic ductal adenocarcinoma (PDAC) Corresponding Author: Daniel J. Renouf, BC Cancer, 600 West 10th Avenue, is defined by the presence of KRAS driver mutations in the vast Vancouver, BC V5Z 4E6, Canada. Phone: 604-877-6000; Fax: 604-877-0585; E-mail: [email protected] majority of cases, and is for the most part highly resistant to molecularly targeted therapeutic strategies. There is, however, a Clin Cancer Res 2019;XX:XX–XX subset of PDACs that are KRAS wild-type. As part of the POG doi: 10.1158/1078-0432.CCR-19-0191 study, we have sequenced tumor biopsies from 47 PDAC patients, 2019 American Association for Cancer Research. 91% of which were characterized by clonal KRAS gain-of-function

www.aacrjournals.org OF1

Jones et al.

reads generated from the tumor sample. Somatic point mutations, Translational Relevance small insertions and deletions (indels), and copy-number altera- Gene fusions involving neuregulin 1 (NRG1) have been tions, present in the tumor DNA but not in the germ line were noted in multiple cancer types and have potential therapeutic identified (7–9). Detection of structural rearrangements was implications. Here, we describe 3 patients with advanced, achieved by merging and annotating structural variants (SV) KRAS wild-type pancreatic ductal adenocarcinoma (PDAC) detected by de novo assembly (Trans-ABySS; ref. 10) and existing who were comprehensively profiled by whole-genome and SV callers—deFUSE (11), DELLY (12), Chimerascan (13), and transcriptome sequencing. All 3 tumors were positive for gene Manta (14)—using MAVIS (15). Fusion and RNA sequencing fusions involving the ERBB3 ligand NRG1. Two of the 3 (RNA-seq) alignment visualization were generated using MAVIS. patients were treated with the HER-family kinase inhibitor Publicly available transcriptome sequencing data from The Can- afatinib and demonstrated a significant and rapid response cer Genome Atlas (TCGA; ref. 16) were used to evaluate the while on therapy. This work adds to a growing body of expression profile of gene transcripts. evidence that NRG1 gene fusions are recurrent, therapeutically actionable genomic events in hepatobiliary/pancreatic can- Mutation signature analysis cers. Based on the clinical outcomes described here, patients The mutation signature profile was determined by classifying with KRAS wild-type PDAC would benefit from routine testing all genomic SNVs into 96 classes based on variant and 30/50 for NRG1 gene fusions. mutation context to obtain a mutation catalog vector as described by Alexandrov and colleagues (17). In order to determine the best fit to a consensus set of 30 mutation signatures (available at http:// cancer.sanger.ac.uk/cosmic/signatures), we performed nonnega- mutations. KRAS wild-type PDAC tumors are thought to be tive least-squares decomposition implemented in the R package dependent upon alternate oncogenic drivers that, though poorly "nnls." understood (5), may provide opportunities for selective interven- tion with appropriate targeted therapies. Here, we present 3 cases Molecular subtyping of KRAS wild-type PDAC that harbor oncogenic NRG1 fusions, Molecular subtyping was performed based on gene-expression with 2 cases demonstrating a significant response when treated signatures detailed in Moffitt and colleagues (18), Bailey and with the HER-family kinase inhibitor afatinib. NRG1 fusions were colleagues (19), and Collisson and colleagues (20). Given the highly expressed, retained the functional EGF-like domain relatively small cohort size, RNA-seq data from 130 TCGA PAAD required for HER-family kinase activation and involved trans- samples were leveraged in order to increase sample size and membrane domain–containing fusion partners. A study pub- enable more robust clustering. Data were normalized using an lished by Heining and colleagues similarly identified 3 NRG1 empirical Bayesian method provided by ComBat (21). Consensus fusion–positive cases in their cohort of 17 PDAC tumors, one of clustering of z-scores (of log10(x þ1)) was performed, where x was which also demonstrated a reduction in tumor size in response to RPKM for POG samples and TPM for TCGA samples (5). Den- treatment with afatinib (6). Together, these observations provide drograms were cut, and branches were labeled to assign subtypes evidence for the recurrent nature of oncogenic NRG1 fusions in according to relative expression levels of subtyping gene sets. KRAS wild-type PDAC and for the beneficial treatment of afatinib in such cases, supporting further investigation of its use in this Sequencing data availability setting. Genomic and transcriptomic data sets have been deposited at the European Genome-phenome Archive (http://www.ebi.ac.uk/ega/) under accession numbers EGAD00001004717 (patient 44), Materials and Methods EGAD00001004718 (patient 45) EGAD00001004716 (patient 46). Patients were enrolled in the POG trial at BC Cancer in Vancouver, British Columbia. Patient biopsy samples received NRG1 fluorescent in situ hybridization protocol whole-genome and transcriptome profiling in order to identify Fluorescent in situ hybridization (FISH) was performed as potentially clinically actionable genomic events. The study was previously described (2) using the Dako Histology FISH Accessory approved by the University of British Columbia Research Kit as per manufacturer's protocol. Slides were scored manually Ethics Committee (REB# H12-00137, H14-00681, H16-00291) using an oil immersion 63 objective, and z-stack images were and was conducted in accordance with international ethical captured using Metasystems software (MetaSystems Group Inc.; guidelines. Written informed consent was obtained from each for details, see Supplementary Methods). patient prior to genomic profiling. Patient identity was anon- ymized, and an identification code was assigned to the case for NRG1 fusion RT-PCR communicating clinically relevant information to physicians. Total RNA was extracted from 6 5 mm scrolls of FFPE Patients consented to potential publication of findings. Raw material with a High Pure FFPET RNA Isolation kit sequence data and downstream analytics were maintained within (Roche). First-strand cDNA was prepared from 200 to 500 ng a secure computing environment. RNA with standard procedures for Superscript IV reverse tran- scriptase and random hexamer oligonucleotides (Thermo Fish- Whole-genome and transcriptome analysis er Scientific). Based on whole-genome and transcriptome WGTA was performed as previously described (2). Briefly, a data, oligonucleotide primers were designed to ATP1B1 exon fresh tumor biopsy and blood sample were collected and 2(FWD:50-CATCGGAACCATCCAAGTGA-30)andexon3 sequenced to a mean redundant depth of coverage of 40 and (FWD:50-TTTCGTCCTAATGATCCCAAGAG-30) and NRG1 exon 80, respectively, and a transcriptome of approximately 200M 2 (RVS:50-CATTCCCATTCTTGAACCACTTG-30) and exon 6

OF2 Clin Cancer Res; 2019 Clinical Cancer Research

NRG1 Gene Fusions in PDAC Are Clinically Actionable

(RVS:50-CAGTGGTGGATGTAGATGTAGC-30). Each case was tor therapy. WGTA was performed on a core needle biopsy from a assessed by standard Platinum Taq polymerase PCR protocol metastatic lesion in the liver. Tumor content was estimated to be (Thermo Fisher Scientific) at 55C annealing with ATP1- 40% by pathology. Analysis of the somatic data revealed 42 B1ex2FWD-NRG1ex2RVS and ATP1B1ex3FWD-NRG1ex6RVS somatic mutations (SNVs and indels; Supplementary Table primer combinations. Positive PCR bands were Sanger S6). The tumor was negative for KRAS mutations, and none of sequenced to confirm the fusion breakpoint (NAPS, Michael the other mutations were determined to be of clinical relevance. Smith Laboratories; for details, see Supplementary Methods). SV analysis revealed a fusion of ATP1B1 (exon 4) with NRG1 (exon 6), which was supported by both the genome and tran- TGCA fusion data review scriptome data (Fig. 1B; Supplementary Table S2). Expression Additional NRG1 gene fusions were identified by querying the analysis of the NRG1 gene revealed the tumor sample from Tumor Fusion Gene Data Portal (http://www.tumorfusions.org; patient 44 was a high expression outlier compared with a cohort ref. 22), which uses the PRADA algorithm (23) to detect fusion of primary pancreatic adenocarcinoma samples, suggesting transcripts. increased expression of NRG1 as a result of the fusion transcript. Alignment of RNA-seq reads to the NRG1 gene demonstrated a significant increase in expression of exons 6 and 7 downstream of Results the predicted breakpoint, indicating the translocation stimulated Genomic and transcriptomic analysis expression of the fusion transcript (Fig. 2B). This patient remains As part of the POG study, metastatic tumors from 47 patients on first-line therapy. with advanced PDAC underwent WGTA. In agreement with Patient 45 is a 59-year-old man with a family history of prostate previous genomic characterization studies of PDAC, clonal KRAS and colon cancers, who presented with stage IV PDAC with gain-of-function mutations were identified in 91% (43/47) of multiple metastases to the liver. No prior germ line or somatic tumor samples (Fig. 1A; Supplementary Table S1). KRAS muta- testing was performed. He was consented and enrolled in the POG tions were not identified in 4 samples by standard somatic single- clinical trial after having received 10 cycles of standard treatment nucleotide variant analysis (patients 44–47; Supplementary Table with oxaliplatin, irinotecan, and 5-FU (FOLFIRINOX), with some S1). Subsequent targeted read alignment of known KRAS gain-of- initial partial response and subsequent progression. He was function hotspots revealed 1 of the 4 initially described KRAS subsequently treated with 3 cycles of gemcitabine with progres- wild-type tumors (patient 47) had a KRAS gain-of-function muta- sion. WGTA analysis performed on a biopsy sample (pathology- tion of low variant allele frequency, indicating a potential sub- estimated tumor content of 40%) from a metastatic lesion from clonal KRAS mutation (1 and 6 sequencing reads supporting a the liver showed 52 somatic mutations (SNVs and indels; Sup- KRAS G12D in the genome and transcriptome, respectively). plementary Table S6), none of which were determined to be Three of the 47 samples were devoid of KRAS mutations based clinically relevant. SV analysis revealed an ATP1B1–NRG1 gene on our standard and targeted SNV calling protocols. fusion that was supported by both the genome and transcriptome SV analysis and annotation from genome and transcriptome data (Fig. 1B; Supplementary Table S2). The breakpoints identi- sequencing data were performed using MAVIS (15). Transloca- fied in patient 45 resulted in fusion of exon 3 of ATP1B1 with exon tions affecting NRG1 were identified in 3 KRAS wild-type 2ofNRG1, and differed from those observed in patient 44. Similar tumor samples (Fig. 1A; Supplementary Table S2). Notably, we to patient 44, NRG1 transcripts were highly expressed in the tumor did not detect oncogenic mutations or SVs in potentially action- sample from patient 45, which was a significant outlier when able alternative drivers including BRAF, ERBB2, ROS1, ALK, compared with the cohort of primary PDAC, as described above NTRK1–3, BRCA1/2, or ATM. Furthermore, recurrent loss-of- (Fig. 2A). In agreement with the fusion breakpoints detected in function mutations and deletions affecting tumor suppressors this sample, aligned RNA-seq reads across the NRG1 gene revealed CDKN2A, TP53, and SMAD4 were absent in tumors positive an increase in expression of exons 2 to 7 (Fig. 2B). ATP1B1–NRG1 for NRG1 fusions (Fig. 1A; Supplementary Tables S1 and S3). fusions were subsequently validated by RT-PCR followed by Additionally, expression-based subtyping revealed that all 3 Sanger sequencing. KRAS wild-type, NRG1 fusion–positive samples were consistent Testing by break-apart FISH did not confirm the presence of with the classic subtype of PDAC (Fig. 1A; Supplementary Table ATP1B1–NRG1 fusions in patient 44 or 45. Upon examination of S4; ref. 18). Somatic SNVs were predominantly C:G>T:A and T: the genome assembly data, additional genomic breakpoints A>C:G transitions, similar to the majority of the cohort (Fig. 1A), downstream of the fusion junction were identified in each case. and were compared with a catalog of previously described muta- An analysis of the structural complexity determined that the tion signatures (http://cancer.sanger.ac.uk/cosmic/signatures). fusions likely arise from the insertion of the C-terminal region Somatic SNVs from all 3 NRG1 fusion–positive tumors were of the NRG1 gene from chromosome 8 into the ATP1B1 locus on most highly attributed to COSMIC mutation signatures 1 and chromosome 1 (Supplementary Fig. S1). 5, which are ubiquitous across cancer types (ref. 24; Supplemen- Patient 46 is a 54-year-old man who has a limited family history tary Table S5). of cancer and presented with stage IV PDAC with metastasis to the liver. The patient was consented and enrolled in the POG NRG1 fusions in advanced PDAC and PanGen clinical trials and received gemcitabine with nab- Patient 44 is a 55-year-old man with a family history of GI paclitaxel as part of a clinical trial. WGTA analysis of the liver cancers (gastric and colorectal cancers), who presented with stage biopsy (pathology-estimated tumor content of 85%) revealed IV PDAC with liver metastases. No prior germ line or somatic 120 somatic mutations (SNVs and indels) including homozygous testing was performed, and he was enrolled on a first-line met- frame-shift mutations affecting ARID1A (p.Q1519fs) and B2M (p. astatic phase II clinical trial of gemcitabine and nab-paclitaxel S53fs) and several variants of unknown significance affecting versus gemcitabine, nab-paclitaxel, and dual-checkpoint inhibi- additional components of the antigen-presenting class I MHC

www.aacrjournals.org Clin Cancer Res; 2019 OF3

Jones et al.

A 600

400

200 Coding mutation count 0 1.00 Substitution 0.75 C:G>A:T C:G>G:C 0.50 C:G>T:A T:A>A:T 0.25 T:A>C:G T:A>G:C 0.00 1 2 3 4 5 6 7 8 9 1011121314151617181920212223242526272829303132333435363738394041424344454647 Variant KRAS Deletion NRG1 Amplification Truncating TP53 Missense CDKN2A Translocation Figure 1. NRG1 fusions detected in KRAS wild- SMAD4 type PDAC. A, Molecular alteration GNAS status of genes recurrently altered in ERBB2 PDAC and gene-expression–based ALK molecular subtyping in the advanced MYC PDAC cohort (n ¼ 47). NRG1 fusions FGFR1 were detected in 3 KRAS wild-type patients. B, Fusion visualization BRCA2 diagrams for NRG1 gene fusions in ATM Subtype KRAS wild-type patient tumors. MSH6 Basal-like NRG1 Classic Exons corresponding to MSH3 na (ENST00000523534), ATP1B1 APP RNF43 Classical (ENST00000367816), and Exo-like (ENST00000346798) incorporated Quasimes Moffitt in the fusion transcripts are Collisson Adex indicated. Reading frame is indicated Immuno Bailey Progen by the black bar below the fusion Squamous transcript model, and EGF-like and transmembrane domains derived Patient 44 from NRG1 and the fusion partners, B respectively, are highlighted by the dark blue and beige boxes. ATP1B1–NRG1

Patient 45

ATP1B1–NRG1

Patient 46

APP–NRG1

EGF-like domain Transmembrane domain

complex, HLA-A (p.V255M and p.D251H) and HLA-B (p.T162K; All 3 NRG1 fusions were predicted to be in-frame and the Supplementary Table S6). The tumor was negative for KRAS gain- predicted fusion structure preserved the EGF-like domain, which of-function mutations. A complex structural rearrangement has previously been shown to be required for NRG1 function resulting in the insertion of exons 6 and 7 of NRG1 in between (Fig. 1B; refs. 25, 26). Moreover, alignment of the RNA-seq reads exons 15 and 16 of APP was detected in both the genome and revealed a striking increase in expression of EGF-like domain- transcriptome (Fig. 1B; Supplementary Table S2). Expression containing exons 6 and 7 in all 3 cases (Fig. 2B). Both fusion analysis indicated the fusion transcript resulted in increased partners, ATP1B1 and APP, encode integral membrane proteins expression of exons 6 and 7 of the NRG1 gene and outlier and donate transmembrane domains to the predicted NRG1 expression compared with the primary PDAC cohort (Fig. 2). fusion proteins (Fig. 1B). Finally, a query of gene fusions detected Similar to patients 44 and 45, insertion of the NRG1 genomic in transcriptome data from TCGA (http://www.tumorfusions. fragment containing exons 6 and 7 into the APP gene were org/; ref. 22) identiﬁed a third ATP1B1–NRG1 in-frame gene predicted to not be detectable by break-apart FISH. fusion in a sample from a PDAC (Table 1). The fusion junction

OF4 Clin Cancer Res; 2019 Clinical Cancer Research

NRG1 Gene Fusions in PDAC Are Clinically Actionable

A pt.46 pt.45 pt.44 Frequency 01020304050

−3 −2 −1 0 12 Figure 2.

Expression of NRG1 fusion transcripts. Log10 (RPKM) A, Histogram of NRG1 expression

[log10(RPKM)] across the primary TCGA pancreatic cohort with expression of NRG1 B fusion–positive PDAC patients indicated by the dashed lines. B, Density of RNA-seq read alignment across the NRG1 transcript (ENST00000523534) for NRG1 fusion– positive PDAC patient tumors.

results in the fusion of exon 3 from ATP1B1 and exon 2 of NRG1, the degree and extent of FDG avidity within supraclavicular, identical to the structure of the gene fusion identified in patient 45 mediastinal, portocaval, and liver had all improved. Repeat (Fig. 1B). CA19-9 after 4 weeks dropped to 7246 (Fig. 3C). Clinically, he appeared much better with significant improvements in pain and Clinical response to afatinib in NRG1 fusion–positive PDAC performance status. He had minor skin rash and minor diarrhea Following progression on gemcitabine, patient 45 obtained related to the afatinib. He had a CT at 3 months after treatment access to afatinib. At the time of starting therapy, he had significant initiation confirming ongoing response. At 5.5 months since ascites and was ECOG performance status 2. His CA19-9 was treatment initiation, he had a repeat CT that demonstrated disease >120,000. A PET/CT scan was performed at baseline prior to progression. initiating therapy (Fig. 3A, left). He initiated treatment at a Patient 46 did not tolerate gemcitabine and nab-paclitaxel and dose of 40 mg daily but was switched to 30 mg during cycle 1 came off treatment after 1 cycle. At the time of starting afatinib, he due to diarrhea. After 4 weeks, a repeat PET/CT was performed had significant progression of disease with PET/CT showing demonstrating a significant response to therapy. It was noted that avidity in the pancreatic head mass, as well as in multiple

Table 1. In-frame ATP1B1–NRG1 gene fusions detected in transcriptome data from TCGA and POG and described in the published literature Cancer type Sample Fusion pair Frame 50 Gene junction 30 Gene junction 50 Exon 30 Exon PDAC POG-Patient 44 ATP1B1–NRG1 In-frame Chr1:169094277/1 Chr8:32585467/1 4 6 PDAC POG-Patient 45 ATP1B1–NRG1 In-frame Chr1:169080736/1 Chr8:32453346/1 3 2 PDAC POG-Patient 46 APP–NRG1 In-frame Chr21:27277336/1 Chr8:32585467/1 15.7 6.16 Chr8:32600251/1 Chr21:27269985/1 CHOL POG-Patient 5(2) ATP1B1–NRG1 In-frame Chr1:169080736/1 Chr8:32453346/1 3 2 PDAC TCGA.3A.A9I5.01A ATP1B1–NRG1 In-frame Chr1:169080736/1 Chr8:32453346/1 3 2 PDAC NCT/DKTK MASTER(6) ATP1B1–NRG1 In-frame Chr1:169080736/1 nd 3 2 BLCA TCGA.FD.A5BR.01A CDK1–NRG1 In-frame Chr10:62539960/1 Chr8:32585467/1 2 6 HNSC TCGA.CQ.5334.01A PDE7A–NRG1 In-frame Chr8:66691955/1 Chr8:32585467/1 3 6 LUAD TCGA.86.7954.01A SDC4–NRG1 In-frame Chr20:43959006/1 Chr8:32585467/1 4 6 LUAD POG-patient 4(2) SDC4–NRG1 In-frame Chr20:43959006/1 Chr8:32585467/1 4 6 Abbreviations: BLCA, bladder urothelial carcinoma; CHOL, cholangiocarcinoma; HNSC, head and neck squamous cell carcinoma; LUAD, lung adenocarcinoma; nd, no data.

www.aacrjournals.org Clin Cancer Res; 2019 OF5

Jones et al.

Pretreatment +4 Weeks afatinib

Figure 3. Clinical response of NRG1 fusion–positive PDAC with afatinib. A, PET/CT scans of patient 45 prior to treatment (left) and after 4 weeks on afatinib (right). B, PET/CT scans Pretreatment +4 Weeks afatinib of patient 46 prior to treatment (left) and after 4 weeks on afatinib (right). C, CA19-9 levels pre- and post-afatinib for patient 45. C Baseline Post-afatinib

100,000

50,000 CA19-9 level

0 012 Time (months)

metastatic lymph nodes and liver metastasis (Fig. 3B, left). CA19- lung cancers and are thought to drive ectopic signaling through 9 levels were within the normal range. He initiated treatment with continuous stimulation of HER-family proteins leading to growth afatinib at a dose of 30 mg daily. After 4 weeks, a repeat PET/CT and proliferation (27, 28). It is, then, not surprising that NRG1 was performed also showing significant response to therapy gene fusions could drive PDAC given that the majority of PDAC is (Fig. 3B, right). There was noted to be resolution of multiple driven by KRAS activation, providing continuous activation of hepatic metastases, and the degree of FDG avidity within the signaling pathways, including MEK, ERK, and PI3K (29). This primary pancreatic mass had also decreased. He tolerated the hypothesis is borne out by the emerging mutual exclusivity of treatment well with good energy and minimal toxicity other than NRG1 gene fusions and KRAS-activating mutations, which sug- minor facial rash and paronychia. Response was confirmed by CT gests an overlapping functions. The absence of potential alterna- imaging performed after 8 weeks following treatment initiation, tive drivers of PDAC in NRG1 fusion–positive tumors provides and a CT scan done 5 months after treatment initiation showed further support for NRG1 fusions as a driver in PDAC. ongoing response. He continues on treatment at the time of Two patients were treated with afatinib as a result of the NRG1 manuscript preparation. fusions detected as part of this study. Both patients demonstrated a significant clinical response when treated with afatinib. This observation is interesting given that a recent publication described Discussion an observable lack of response in some lung cancers positive for In this report, we describe ATP1B1–NRG1 gene fusions in 2 of NRG1 fusions (4). This perhaps suggests that the fusion partner or the 47 PDAC samples sequenced as part of the ongoing POG trial etiology has an influence on how well a given tumor will respond. at our institute. Within this cohort, 3 PDAC samples were KRAS Alternatively, the genomic context of the gene fusion may be the wild-type and harbored NRG1 fusions. A fourth was identified in a relevant factor indicating the importance of comprehensive geno- review of publicly available transcriptome, and 3 additional cases mic profiling to determine if the gene fusions detected are likely to were recently described in independent study data (6). Together, be the sole driver of the disease. Further work is required to explore these results indicate that NRG1 fusions occur in a small propor- these observations. tion of PDAC patients, potentially defining a new subtype of KRAS Patient 45 showed disease progression after 5.5 months on wild-type PDAC. treatment, a response to afatinib broadly equivalent to that seen in NRG1 activates the ERBB3 (HER3) receptor, which then forms a an unselected population of patients with advanced NSCLC (30). heterodimer with other HER-family receptors, regulating down- This observation demonstrates the likelihood of resistance stream signaling pathways. NRG1 gene fusions are prevalent in mechanisms arising in pretreated NRG1 fusion–positive PDAC.

OF6 Clin Cancer Res; 2019 Clinical Cancer Research

NRG1 Gene Fusions in PDAC Are Clinically Actionable

Upregulation of NRG1 is a well-documented mechanism of and Taiho. No potential conflicts of interest were disclosed by the other parallel pathway activation and resistance in HER2-positive breast authors. cancer models (31, 32) and ALK-positive lung cancer (33, 34). Furthermore, it has been reported that PDAC cells treated with an Authors' Contributions ERK-specific inhibitor upregulated the parallel PI3K–AKT path- Conception and design: M.R. Jones, H.J. Lim, J. Laskin, D.F. Schaeffer, way through activating HER-family proteins (35). It is therefore S.J.M. Jones, D.J. Renouf – Development of methodology: E. Pleasance, H.J. Lim, S. Yip, A.J. Mungall, perhaps likely that resistance mechanisms in HER-family driven J. Laskin, S.J.M. Jones, D.J. Renouf PDAC will emerge from the acquisition of genomic alterations Acquisition of data (provided animals, acquired and managed patients, that activate parallel signaling pathways. provided facilities, etc.): M.R. Jones, M.K.C. Lee, J. Ho, G. Jang, With respect to possible therapeutic strategies, a recent study J.M. Karasinska, J.P. McGhie, S. Gill, H.J. Lim, H.L. Wong, T. Ng, S. Yip, has demonstrated that concurrent inhibition or HER-family A.J. Mungall, R.A. Moore, J.J. Knox, S. Gallinger, J. Laskin, M.A. Marra, kinases synergizes with an ERK-specific inhibitor in suppressing D.J. Renouf in vitro in vivo Analysis and interpretation of data (e.g., statistical analysis, biostatistics, PDAC cell growth and (35). This provides a computational analysis): M.R. Jones, L.M. Williamson, J.T. Topham, rationale for testing combinations of targeted therapies in M.K.C. Lee, R.E. Denroche, E. Pleasance, Y. Shen, H.J. Lim, M.J. Moore, preclinical models, as has been demonstrated for cancers trea- H.L. Wong, T. Ng, W. Zhang, S. Sadeghi, C. Reisle, K.L. Mungall, Y. Ma, ted with BRAF inhibitors (36, 37). Also, given that NRG1 is the J. Laskin, M.A. Marra, S.J.M. Jones, D.J. Renouf primary ligand of ERBB3 (38), there is significant interest in Writing, review, and/or revision of the manuscript: M.R. Jones, targeting ERBB3 directly. A recent study using breast cancer cell L.M. Williamson, J.T. Topham, M.K.C. Lee, A. Goytain, S. Gill, H.J. Lim, NRG1 NRG1 M.J. Moore, H.L. Wong, T. Ng, S. Yip, C. Reisle, A.J. Mungall, J.J. Knox, lines harboring an gene fusion and a model of S. Gallinger, J. Laskin, M.A. Marra, D.F. Schaeffer, S.J.M. Jones, D.J. Renouf – fusion positive ovarian cancer revealed potential complexities Administrative, technical, or material support (i.e., reporting or organizing in responses to broad versus targeted HER-family kinase inhi- data, constructing databases): A. Goytain, H.J. Lim, M.J. Moore, H.L. Wong, bitors in NRG1 fusion–positive disease (4). However, estab- A.J. Mungall, Y. Ma, S. Gallinger lishing an NRG1-positive model of PDAC will be instrumental Study supervision: J. Laskin, M.A. Marra, D.F. Schaeffer, D.J. Renouf in investigating appropriate treatments strategies for this etiology. To date, no ERBB3-targeted therapy has been approved for Acknowledgments cancer treatment, though monoclonal antibodies are under The authors gratefully acknowledge the participation of their patients and families, the POG and PanGen team, and the generous support of the BC clinical evaluation (39, 40) and the number of phase I and Cancer Foundation and Genome British Columbia. The results published phase II clinical trials continues to grow. However, given the here are in part based upon data generated by The Cancer Genome Atlas rarity and structural complexity of NRG-1 fusions, developing managed by the NCI and NHGRI (http://cancergenome.nih.gov). M.R. Jones targeted strategies to reliably detect these genomic events will had full access to all the data in the study and takes responsibility for the be necessary to facilitate the appropriate interpretation of integrity of the data and the accuracy of the data analysis. M.R. Jones, L.M. clinical trial outcomes. Williamson (BC Cancer, Canada's Michael Smith Genome Sciences Centre), and J.T. Topham (Pancreas Centre BC, Vancouver, Canada) conducted and Finally, in addition to a previously described SDC4–NRG1 NRG1 are responsible for the data analysis. Thank you to Dr. Daniel F. Worsley and gene fusion in a case of lung cancer, 2 additional in-frame Dr. R. Petter Tonseth for their help in obtaining the radiologic images. The gene fusions were identified in transcriptome data from TCGA: authors acknowledge contributions toward equipment and infrastructure CDK1–NRG1 in a bladder urothelial carcinoma and PDE7A– from Genome Canada and Genome British Columbia (projects 202SEQ, NRG1 in a head and neck squamous cell carcinoma (Table 1). 212SEQ, 12002), Canada Foundation for Innovation (projects 20070, Though the evidence is currently limited, this finding suggests that 30198, 30981, 33408), and the BC Knowledge Development Fund. This research was generously supported through unrestricted philanthropic dona- broad screening for NRG1 gene fusions across different cancer tions received through the BC Cancer Foundation, as well as funding types may reveal additional potentially clinically actionable onco- provided by the Terry Fox Research Institute, Pancreatic Cancer Canada, genic events that can be tested for their therapeutic relevance in a and Genome British Columbia (project B20POG). M.A. Marra acknowledges clinical or preclinical setting. infrastructure investments from the Canada Foundation for Innovation and the support of the Canada Research Chairs and CIHR Foundation (FDN- Disclosure of Potential Conflicts of Interest 143288) programs. H.J. Lim is a consultant/advisory board member for Bristol-Myers Squibb, Roche, Ipsen, Eisai, Taiho, and Amgen. S. Yip is a consultant/advisory The costs of publication of this article were defrayed in part by the board member for Bayer, Pfizer,andRoche.J.J.Knoxreportsreceivingother payment of page charges. This article must therefore be hereby marked advertisement commercial research support from Merck, and is a consultant/advisory board in accordance with 18 U.S.C. Section 1734 solely to indicate member for Merck and Bristol-Myers Squibb. J. Laskin reports receiving this fact. other remuneration from Roche Canada and BI Canada. D.J. Renouf reports receiving speakers bureau honoraria from Servier and Celgene, and Received January 16, 2019; revised March 7, 2019; accepted April 15, 2019; is a consultant/advisory board member for Servier, Celgene, Ipsen, Bayer, published first May 8, 2019.

References 1. Gay ND, Wang Y, Beadling C, Warrick A, Neff T, Corless CL, et al. Durable 3. Cheema PK, Doherty M, Tsao M-S. A case of invasive mucinous pulmonary response to afatinib in lung adenocarcinoma harboring NRG1 gene adenocarcinoma with a CD74-NRG1 fusion protein targeted with afatinib. fusions. J Thorac Oncol 2017;12:e107–10. J Thorac Oncol 2017;12:e200–2. 2. Jones MR, Lim H, Shen Y, Pleasance E, Ch'ng C, Reisle C, et al. Successful 4. Drilon A, Somwar R, Mangatt BP, Edgren H, Desmeules P, Ruusulehto A, targeting of the NRG1 pathway indicates novel treatment strategy for et al. Response to ERBB3-directed targeted therapy in NRG1-rearranged metastatic cancer. Ann Oncol 2017;28:3092–7. cancers. Cancer Discov 2018;8:686–95.

www.aacrjournals.org Clin Cancer Res; 2019 OF7

Jones et al.

5. Cancer Genome Atlas Research Network. Integrated genomic characteri- 23. Torres-García W, Zheng S, Sivachenko A, Vegesna R, Wang Q, Yao R, et al. zation of pancreatic ductal adenocarcinoma. Cancer Cell 2017;32: PRADA: pipeline for RNA sequencing data analysis. Bioinforma Oxf Engl 185–203.e13. 2014;30:2224–6. 6. Heining C, Horak P, Uhrig S, Codo PL, Klink B, Hutter B, et al. NRG1 24. Alexandrov LB, Nik-Zainal S, Wedge DC, Aparicio SA, Behjati S, Biankin AV, fusions in KRAS wild-type pancreatic cancer. Cancer Discov 2018;8: et al. Signatures of mutational processes in human cancer. Nature 2013; 1087–95. 500:415–21. 7. Saunders CT, Wong WS, Swamy S, Becq J, Murray LJ, Cheetham RK. Strelka: 25. Jung Y, Yong S, Kim P, Lee HY, Jung Y, Keum J, et al. VAMP2-NRG1 fusion accurate somatic small-variant calling from sequenced tumor-normal gene is a novel oncogenic driver of non-small-cell lung adenocarcinoma. sample pairs. Bioinforma Oxf Engl 2012;28:1811–7. J Thorac Oncol 2015;10:1107–11. 8. Ding J, Bashashati A, Roth A, Oloumi A, Tse K, Zeng T, et al. Feature-based 26. Guo R, Zhang Y, Li X, Song X, Li D, Zhao Y. Discovery of ERBB3 inhibitors classifiers for somatic mutation detection in tumour-normal paired for non-small cell lung cancer (NSCLC) via virtual screening. J Mol Model sequencing data. Bioinforma Oxf Engl 2012;28:167–75. 2016;22:135. 9. Ha G, Roth A, Lai D, Bashashati A, Ding J, Goya R, et al. Integrative analysis 27. Saito M, Shiraishi K, Kunitoh H, Takenoshita S, Yokota J, Kohno T. Gene of genome-wide loss of heterozygosity and monoallelic expression at aberrations for precision medicine against lung adenocarcinoma. nucleotide resolution reveals disrupted pathways in triple-negative breast Cancer Sci 2016;107:713–20. cancer. Genome Res 2012;22:1995–2007. 28. Jeong H, Kim J, Lee Y, Seo JH, Hong SR, Kim A. Neuregulin-1 induces cancer 10. Robertson G, Schein J, Chiu R, Corbett R, Field M, Jackman SD, et al. De stem cell characteristics in breast cancer cell lines. Oncol Rep 2014;32: novo assembly and analysis of RNA-seq data. Nat Methods 2010;7: 1218–24. 909–12. 29. Polireddy K, Chen Q. Cancer of the pancreas: molecular pathways and 11. McPherson A, Hormozdiari F, Zayed A, Giuliany R, Ha G, Sun MGF, et al. current advancement in treatment. J Cancer 2016;7:1497–514. deFuse: an algorithm for gene fusion discovery in tumor RNA-Seq data. 30. Ezeife DA, Melosky B, Tudor R, Lin S, Lau A, Panzarella T, et al. Afatinib in PLoS Comput Biol 2011;7:e1001138. advanced pretreated non-small-cell lung cancer—a Canadian experience. 12. Rausch T, Zichner T, Schlattl A, Stutz€ AM, Benes V, Korbel JO. DELLY: Curr Oncol Tor Ont 2018;25:e385–90. structural variant discovery by integrated paired-end and split-read anal- 31. Xia W, Petricoin EF 3rd, Zhao S, Liu L, Osada T, Cheng Q, et al. An ysis. Bioinforma Oxf Engl 2012;28:i333–9. heregulin-EGFR-HER3 autocrine signaling axis can mediate acquired lapa- 13. Iyer MK, Chinnaiyan AM, Maher CA. ChimeraScan: a tool for identifying tinib resistance in HER2þ breast cancer models. Breast Cancer Res 2013;15: chimeric transcription in sequencing data. Bioinforma Oxf Engl 2011;27: R85. 2903–4. 32. Prentice LM, Shadeo A, Lestou VS, Miller MA, deLeeuw RJ, Makretsov N, 14. Chen X, Schulz-Trieglaff O, Shaw R, Barnes B, Schlesinger F, K€allberg M, et al. NRG1 gene rearrangements in clinical breast cancer: identification of et al. Manta: rapid detection of structural variants and indels for germline an adjacent novel amplicon associated with poor prognosis. Oncogene and cancer sequencing applications. Bioinforma Oxf Engl 2016;32: 2005;24:7281–9. 1220–2. 33. Dong X, Fernandez-Salas E, Li E, Wang S. Elucidation of resistance mechan- 15. Reisle C, Mungall KL, Choo C, Paulino D, Bleile DW, Muhammadzadeh A, isms to second-generation ALK inhibitors alectinib and ceritinib in non- et al. MAVIS: merging, annotation, validation, and illustration of structural small cell lung cancer cells. Neoplasia 2016;18:162–71. variants. Bioinforma Oxf Engl 2019;35:515–7. 34. Kimura M, Endo H, Inoue T, Nishino K, Uchida J, Kumagai T, et al. Analysis 16. Cancer Genome Atlas Research Network, Weinstein JN, Collisson EA, Mills of ERBB ligand-induced resistance mechanism to crizotinib by primary GB, Shaw KR, Ozenberger BA, et al. The cancer genome atlas pan-cancer culture of lung adenocarcinoma with EML4-ALK fusion gene. J Thorac analysis project. Nat Genet 2013;45:1113–20. Oncol 2015;10:527–30. 17. Alexandrov LB, Nik-Zainal S, Wedge DC, Campbell PJ, Stratton MR. 35. Jiang H, Xu M, Li L, Grierson P, Dodhiawala P, Highkin M, et al. Concurrent Deciphering signatures of mutational processes operative in human cancer. HER or PI3K inhibition potentiates the antitumor effect of the ERK Cell Rep 2013;3:246–59. inhibitor ulixertinib in preclinical pancreatic cancer models. Mol Cancer 18. MoffittRA,MarayatiR,FlateEL,VolmarKE,LoezaSG,HoadleyKA, Ther 2018;17:2144–55. et al. Virtual microdissection identifies distinct tumor- and stroma- 36. Xue Y, Martelotto L, Baslan T, Vides A, Solomon M, Mai TT, et al. An specific subtypes of pancreatic ductal adenocarcinoma. Nat Genet 2015; approach to suppress the evolution of resistance in BRAFV600E-mutant 47:1168–78. cancer. Nat Med 2017;23:929–37. 19. Bailey P, Chang DK, Nones K, Johns AL, Patch AM, Gingras MC, et al. 37. Firestone AJ, Settleman J. A three-drug combination to treat BRAF-mutant Genomic analyses identify molecular subtypes of pancreatic cancer. Nature cancers. Nat Med 2017;23:913–4. 2016;531:47–52. 38. Mujoo K, Choi B-K, Huang Z, Zhang N, An Z. Regulation of ERBB3/HER3 20. Collisson EA, Sadanandam A, Olson P, Gibb WJ, Truitt M, Gu S, et al. signaling in cancer. Oncotarget 2014;5:10222–36. Subtypes of pancreatic ductal adenocarcinoma and their differing 39. Shimizu T, Yonesaka K, Hayashi H, Iwasa T, Haratani K, Yamada H, et al. responses to therapy. Nat Med 2011;17:500–3. Phase 1 study of new formulation of patritumab (U3-1287) process 2, a 21. Johnson WE, Li C, Rabinovic A. Adjusting batch effects in microarray fully human anti-HER3 monoclonal antibody in combination with erlo- expression data using empirical Bayes methods. Biostat Oxf Engl 2007;8: tinib in Japanese patients with advanced non-small cell lung cancer. 118–27. Cancer Chemother Pharmacol 2017;79:489–95. 22. Hu X, Wang Q, Tang M, Barthel F, Amin S, Yoshihara K, et al. 40. Karachaliou N, Lazzari C, Verlicchi A, Sosa AE, Rosell R. HER3 as a TumorFusions: an integrative resource for cancer-associated transcript therapeutic target in cancer. BioDrugs Clin Immunother Biopharm Gene fusions. Nucleic Acids Res 2018;46:D1144–9. Ther 2017;31:63–73.

OF8 Clin Cancer Res; 2019 Clinical Cancer Research

NRG1 Gene Fusions Are Recurrent, Clinically Actionable Gene Rearrangements in KRAS Wild-Type Pancreatic Ductal Adenocarcinoma

Martin R. Jones, Laura M. Williamson, James T. Topham, et al.

Clin Cancer Res Published OnlineFirst May 8, 2019.

Updated version Access the most recent version of this article at: doi:10.1158/1078-0432.CCR-19-0191

Supplementary Access the most recent supplemental material at: Material http://clincancerres.aacrjournals.org/content/suppl/2019/05/08/1078-0432.CCR-19-0191.DC1

E-mail alerts Sign up to receive free email-alerts related to this article or journal.

Reprints and To order reprints of this article or to subscribe to the journal, contact the AACR Publications Subscriptions Department at [email protected].

Permissions To request permission to re-use all or part of this article, use this link http://clincancerres.aacrjournals.org/content/early/2019/06/03/1078-0432.CCR-19-0191. Click on "Request Permissions" which will take you to the Copyright Clearance Center's (CCC) Rightslink site.