Clinicogenomic Analysis of FGFR2-Rearranged Cholangiocarcinoma Identifies Correlates of Response and Mechanisms of Resistance to Pemigatinib

Published OnlineFirst November 20, 2020; DOI: 10.1158/2159-8290.CD-20-0766

Research Article

Clinicogenomic Analysis of FGFR2- Rearranged Cholangiocarcinoma Identifies Correlates of Response and Mechanisms of Resistance to Pemigatinib

Ian M. Silverman1, Antoine Hollebecque2, Luc Friboulet2, Sherry Owens1, Robert C. Newton1, Huiling Zhen3, Luis Féliz4, Camilla Zecchetto5, Davide Melisi5, and Timothy C. Burn1

abstract Pemigatinib, a selective FGFR1–3 inhibitor, has demonstrated antitumor activity in FIGHT-202, a phase II study in patients with cholangiocarcinoma harboring FGFR2 fusions/rearrangements, and has gained regulatory approval in the United States. Eligibility for FIGHT- 202 was assessed using genomic profiling; here, these data were utilized to characterize the genomic landscape of cholangiocarcinoma and to uncover unique molecular features of patients harboring FGFR2 rearrangements. The results highlight the high percentage of patients with cholangiocarcinoma harboring potentially actionable genomic alterations and the diversity in gene partners that rearrange with FGFR2. Clinicogenomic analysis of pemigatinib-treated patients identified mechanisms of primary and acquired resistance. Genomic subsets of patients with other potentially actionable FGF/ FGFR alterations were also identified. Our study provides a framework for molecularly guided clinical trials and underscores the importance of genomic profiling to enable a deeper understanding of the molecular basis for response and nonresponse to targeted therapy.

SIGNIFICANCE: We utilized genomic profiling data from FIGHT-202 to gain insights into the genomic landscape of cholangiocarcinoma, to understand the molecular diversity of patients with FGFR2 fusions or rearrangements, and to interrogate the clinicogenomics of patients treated with pemigatinib. Our study highlights the utility of genomic profiling in clinical trials.

INTRODUCTION line chemotherapy shows limited efficacy in patients with advanced biliary tract cancer (9–11). Cholangiocarcinoma is the most common primary malig- Genomic profiling, based on next-generation sequencing nancy of the bile duct and accounts for 3% of all gastrointes- (NGS) of a panel of genes known to be altered in cancer, tinal tumors (1, 2). Cholangiocarcinoma comprises a group allows for the simultaneous detection of numerous genomic of heterogeneous tumors categorized as intrahepatic and alteration (GA) types, including mutations, copy-number extrahepatic (perihilar and distal), based on biliary tract loca- alterations, and fusions or rearrangements (12). Therefore, tion (2). Incidence and mortality rates of cholangiocarcinoma this technique provides a powerful basis for guiding choice have increased over the past decades, most notably for intra- of targeted therapy, improved diagnosis, and identification hepatic cholangiocarcinoma (3–5). of prognostic and predictive biomarkers. Genomic analysis of The prognosis of patients with cholangiocarcinoma is patients with cholangiocarcinoma has revealed alterations poor; surgery is the only potentially curative therapeutic of targetable oncogenes in almost 50% of patients (13), with option (6). However, as most patients present with advanced recurrent alterations in IDH1 and FGFR2 occurring almost disease, only approximately one third of newly diagnosed exclusively in patients with intrahepatic cholangiocarcinoma patients qualify for surgery. Among patients who are quali- compared with extrahepatic cholangiocarcinoma (13–15). fied to undergo potentially curative resection, most ∼( 76%) Specifically,FGFR2 fusions or rearrangements are observed in will experience a relapse within 2 years (7). For patients with 10% to 16% of patients with intrahepatic cholangiocarcinoma locally advanced or metastatic disease, the standard-of-care (16–18). FGFR2 GAs, including activating point mutations, first-line systemic treatment is chemotherapy with gemcit- fusions, and rearrangements, are known oncogenic drivers abine plus cisplatin (8). There is no established standard of and provide a molecular signature to identify patients who care following first-line chemotherapy failure, and second- may benefit from inhibition of FGFR2 tyrosine kinase activity (19, 20). Therefore, NGS-based assays are used to comprehen- sively identify patients with cholangiocarcinoma who may benefit from targeted therapies. 1Incyte Research Institute, Wilmington, Delaware. 2Institut Gustave Roussy, Pemigatinib is the first targeted therapeutic agent approved 3 4 Villejuif, France. Incyte Corporation, Wilmington, Delaware. Incyte Bio- in the United States for use in cholangiocarcinoma with sciences International Sàrl, Morges, Switzerland. 5Digestive Molecular Clinical Oncology Research Unit, Section of Medical Oncology, Università FGFR2 fusions or rearrangements. Pemigatinib is a selec- degli Studi di Verona, Verona, Italy. tive, potent, oral, competitive inhibitor of FGFR1, 2, and Note: Supplementary data for this article are available at Cancer Discovery 3 that inhibits receptor autophosphorylation and subse- Online (http://cancerdiscovery.aacrjournals.org/). quent activation of FGF/FGFR-mediated signaling networks, Corresponding Author: Timothy C. Burn, Incyte Research Institute, 1801 leading to an inhibition of tumor cell growth in FGFR- Augustine Cut-Off, Wilmington, DE 19803. Phone: 302-498-6787; E-mail: driven cancers (21). The FIbroblast Growth factor receptor [email protected] inhibitor in oncology and Hematology Trial (FIGHT-202; Cancer Discov 2021;11:1–14 NCT02924376) is a phase II, multicenter, open-label study doi: 10.1158/2159-8290.CD-20-0766 of pemigatinib monotherapy in previously treated patients ©2020 American Association for Cancer Research. with locally advanced, metastatic, or surgically unresectable

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RESEARCH ARTICLE Silverman et al. cholangiocarcinoma, including patients with FGFR2 fusions rearrangement partners in cholangiocarcinoma that have or rearrangements (22). In this study, patients were initially been reported by multiple groups (13, 25, 26). According to prescreened prior to enrollment for FGF/FGFR alterations FoundationOne, FGFR2 rearrangements are further defined including amplifications, mutations, fusions, or rearrange- as fusions (i) if the genomic breakpoint is within the intron ments. The primary analysis of 107 patients included only 17 or exon 18 hotspot and (ii) if the fusion gene partner is patients harboring FGFR2 fusions or rearrangements. In either a previously described fusion partner or a novel gene these patients, pemigatinib monotherapy resulted in an inde- partner predicted to be an in-frame fusion with FGFR2. Other pendent centrally confirmed objective response rate (ORR) reported FGFR2 rearrangements include those with genomic of 35.5% and a disease control rate of 82%. With a median breakpoint within the FGFR2 intron 17 or exon 18 hotspot follow-up of 15.4 months, responses were durable, with a and with either (i) a novel partner gene predicted to be out median duration of response of 7.5 [95% confidence inter- of frame or out of strand with FGFR2, or (ii) no identifiable val (CI), 5.7−14.5] months. Median progression-free survival partner gene (designated as intron 17 rearrangement or part- (PFS) was 6.9 (95% CI, 6.2–9.6) months. ner N/A). Therefore, FGFR2 fusions are a subset of FGFR2 There is significant molecular diversity ofFGFR2 fusions rearrangements and collectively are referred to as FGFR2 and rearrangements in patients with intrahepatic cholan- rearrangements. giocarcinoma including a large number of partner genes that rearrange with FGFR2 (13, 17, 18, 23). Therefore, assays that GAs in Cholangiocarcinoma are capable of detecting known and novel FGFR2 fusions or In total, from 1,206 patients we identified 5,547 GAs in 335 rearrangements are necessary to ensure the comprehensive genes, accounting for a mean of 4.6 GAs per patient. Short characterization of tumors, which then enhances the likeli- variants were the most frequent GAs (3,424; 2.8 alterations/ hood of identifying all patients who may respond to FGFR2- patient), followed by copy-number alterations (1,676; 1.39 targeted therapies. It is not yet known whether the FGFR2 alterations/patient) and rearrangements (450; 0.37 altera- partner gene has an impact on response or survival associated tions/patient). The most frequently altered genes were TP53 with FGFR inhibitor treatment. In addition, the question (40.0%), CDKN2A (29.0%), KRAS (22.6%), CDKN2B (19.7%), arises of whether FGFR2 rearrangements co-occur with other ARID1A (16.0%), SMAD4 (11.7%), IDH1 (10.2%), and BAP1 GAs, and whether this may also affect response to therapy. (10.2%; Fig. 1A). Potential clinically actionable alterations, Using the genomic profiling and clinical results from defined as oncogenic driver alterations [including microsat- patients prescreened and enrolled in the FIGHT-202 trial, ellite instability–high (MSI-H) and high tumor mutational this study explores: (i) overall genomic landscape of chol- burden (TMB; >20 mutations per megabase)] with matched angiocarcinoma; (ii) unique genomic features characteristic therapeutic agents either under investigation or approved of FGFR2-rearranged cholangiocarcinoma; (iii) genomic cor- in other tumor types, were identified in 44.5% of patients relates of response, including correlations with FGFR2 rear- (Fig. 1B–D). These included IDH1 mutations (10.2%), ERBB2 rangement partner and co-occurring GAs; (iv) response to mutations and amplifications (8.0%),FGFR2 mutations or pemigatinib in patients without FGFR2 rearrangements; and rearrangements (7.1%), PIK3CA mutations (7.0%), and BRAF (v) acquired resistance to pemigatinib. mutations or rearrangements (4.7%). Other less common oncogenic alterations (in order of decreasing frequency) were in NRAS, IDH2, EGFR, KRAS (G12C mutations only), MET, RESULTS FGFR3, FGFR1, RET, JAK2, ALK, and ROS1 (Fig. 1B). MSI-H To identify patients for enrollment in FIGHT-202, 1,206 and high TMB level were identified in 0.7% and 1.2% of patients with cholangiocarcinoma from any anatomic loca- patients, respectively. No patient with a clinically actionable tion (however, in some instances, noncholangiocarcinoma oncogenic driver alteration had co-occurring MSI-H status biliary tract cancers may have been sent for analysis), based compared with 9 patients in the nonactionable cohort (P = in the United States (n = 138), Europe (n = 569), and Middle 0.0042; Fisher exact test; Fig. 1C). High TMB was observed in East and Asia (rest of the world; n = 499), were prescreened 4 patients with actionable oncogenic driver alterations, com- using the FoundationOne assay (Foundation Medicine Inc.) pared with 10 patients without actionable oncogenic driver for FGF/FGFR alterations prior to eligibility criteria assess- alterations (P = 0.1789; Fisher exact test; Fig. 1D). Although ment (Supplementary Table S1). We did not capture the MSI-H and high TMB are highly correlated (27), 4 patients precise anatomic location of prescreening samples, for exam- with actionable oncogenic driver alterations, including BRAF ple, whether the primary lesion was intrahepatic or extrahe- p.D594N, ERBB2 amplification,FGFR3–TK2 rearrangement, patic. Furthermore, this prescreening population does not and PIK3CA p.E545K, had high TMB in the absence of MSI-H include 85 patients with an existing FoundationOne report status. No patients with an FGFR2 fusion or rearrangement or an FGF/FGFR status report derived from a local assessment had high TMB or MSI-H status. with retrospective central confirmation using Foundation- We also explored the co-alteration and mutual exclusiv- One. The DNA-based, targeted NGS assay detects multiple ity patterns of GAs in the prescreening cohort (Fig. 1E and alteration classes in up to 404 genes including those encod- Supplementary Table S2). The most significant relationship ing components of the FGFR signaling pathway: FGFR1–4, between gene pairs was the co-occurrence of alterations in FGF3/4/6/10/12/14/19/23, and the FGFR substrate 2 (FRS2) FGFR2 and BAP1 (OR, 9.6; q value, 1.4e-14). TP53 was sig- adapter protein (24). FoundationOne detects rearrangements nificantly co-altered withERBB2 (OR, 5.4; q value, 1.7e-10), in select genes, including FGFR1–3, with both known and CCNE1 (OR, 10.5; q value, 3.4e-6), and SMAD4 (OR, 2.7; q novel partners. This is critical due to the diversity of FGFR2 value, 6.5e-5). TP53 was mutually exclusive with BAP1 (OR,

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A B E 10.0% BAP1 : FGFR2 30% Multiple 20% Alteration Alteration Multiple 7.5% Multiple 10% Amplification 10 TP53 : ERBB2 Rearrangement Fusion TP53 : BAP1 0% 5.0% Missense value ) TP53 : IDH1 Copy-number alteration q ( 30% Frequency KRAS : FGFR2 TP53 : CCNE1 Alteration 10 20% 2.5% 5 TP53 : FGFR2

Amplification Lo g SMAD4 : IDH1 TP53 : SMAD4 10% Loss − SMAD4 : BAP1 IDH1 : PBRM1 TP53 : IDH2 BAP1 : NRAS 0.0% 0% SMAD4 : ERBB2 30% Rearrangement 0 ALK RET MET IDH1 IDH2 JAK2 BRAF KRAS ROS1 Alteration NRAS EGFR −4 0 4 FGFR2 FGFR3 FGFR1 20% ERBB2 Fusion Duplication PIK3CA Truncation Rearrangement Log (OR) 10% Deletion Frequency 00.02 0.04 0.06 0% C Short-variant Alterations, n MSI-HMSS Total 30% Alteration Frequency Actionable 0463 463 Missense Nonsense 20% Frame-shift In-frame indel 10% Promoter Splice-site Nonactionable 9506 515

0% Total 9969 978

Total 40% D 30% TMB 20% Alterations, n TMB-H intermediate/low TMB low Total

10% Actionable 498 360 462 0% Nonactionable 10 124 379 513 L3 H1 APC ATM ID TP53 ML BAP1 FRS2 TERT BRAF PTEN KRAS MDM2 ARID 2 STK1 1 FGF1 9 ERBB2 FGFR 2 RBM1 0 PBRM1 CCND 1 SMAD4 PIK3CA ARID1 A Total CDKN2B 14 222 739 975 CDKN2A

Figure 1. Genomic profiling in FIGHT-202. A, Bar graph indicating 25 most frequently altered genes in FIGHT-202 prescreening split by variant type. Colors indicate alteration type (see legend). B, Frequency of clinically actionable alterations. For KRAS, only p.G12C was considered a potentially targetable oncogenic alteration. C and D, Contingency tables for the relationship between (C) MSI status or (D) TMB level and presence of an actionable alteration. E, Co-alteration and mutual exclusivity analysis. Only pairs of alterations with q value < 0.01 and a combined population frequency of >10% were visualized. ORs of 0 were corrected to 0.001 to allow for visualization. MSS, microsatellite stable; TMB-H, TMB-high.

0.2; q value, 3.2e-9), IDH1 (OR, 0.2; q value, 2.3e-8), FGFR2 By combining the FGFR2-rearranged patients identified (OR, 0.2; q value, 6.8e-6), and IDH2 (OR, 0.04; q value, 1.0e-3). through prescreening with FoundationOne and patients with FGFR2 was also significantly mutually exclusive withKRAS a preexisting FoundationOne report or those identified by (OR, 0.04; q value, 5.1e-6), but not IDH1 or IDH2. Across local genomic testing that were confirmed by FoundationOne, the prescreening and enrolled patient cohorts (described in we obtained genomic data on 138 patients with FGFR2 rear- the following section), 8 patients had both activating FGFR2 rangements. In total, we identified 140FGFR2 rearrangements alterations (seven rearrangements and one mutation) and (2 patients each had 2 FGFR2 rearrangements), 113 of which IDH1-activating mutations (p.R132C/G/H/L/S; Supplemen- were predicted to be fusions by FoundationOne (Supplemen- tary Fig. S1 and Supplementary Table S3). tary Table S5; see Methods). We observed 63 unique FGFR2 rearrangement partner genes; the most frequent rearrangement Genomic Landscape of FGFR2-Rearranged found was the FGFR2–BICC1 fusion, accounting for 27.9% of Cholangiocarcinoma FGFR2 rearrangements (Fig. 2B and Supplementary Table S5). In the prescreening cohort, we identified 74 patients (6.1%) Other recurrent FGFR2 partner genes included KIAA1217 (3.6%), with FGFR2 rearrangements (Supplementary Table S1). The TACC2 (2.9%), CCDC6 (2.9%), and AHCYL1 (2.9%). The second frequency of FGFR2 rearrangements varied among regions. In most frequent FGFR2 rearrangement identified wasFGFR2 -N/A the United States, 21 patients (15.2%) were positive, whereas (9.3%; N/A refers to rearrangements that occur in FGFR2 intron 42 (7.4%) were positive in Europe and 11 (2.2%) were identified 17 or exon 18 fused to an intergenic region; see Methods). Strik- in the rest of the world. The frequencies of other GAs also var- ingly, 32.9% of patients had a rearrangement partner that was ied among regions, with higher rates of TP53, CDKN2A, and unique to that patient and not shared with any other patient, KRAS and lower rates of BAP1 and PBRM1 observed in the rest whereas 15.7% of patients had a rearrangement partner that was of the world versus Europe and the United States (Supplemen- shared with just one other patient. FGFR2 rearrangements most tary Fig. S2 and Supplementary Table S4). Among patients frequently occurred intrachromosomally on chromosome 10 identified asFGFR2 rearrangement–positive, 43 were enrolled (52.9%). However, interchromosomal rearrangements occurred on the study into the primary analysis cohort (cohort A). An with an additional 17 chromosomes, most commonly chromo- additional 64 patients were enrolled into cohort A based on a some 1 (7.9%) and chromosome 12 (6.4%; Fig. 2C). A total of preexisting FoundationOne report or other local laboratory 15 patients had 17 FGFR2 mutations in the absence of FGFR2 test (report-in-hand; Fig. 2A). All patients enrolled in cohort A fusions or rearrangements, most commonly p.C382R (n = 7) with local testing were confirmed by FoundationOne. and p.P253R (n = 3; Supplementary Fig. S3).

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AB BICC1 Local tumor N/A Prescreened for KIAA1217 profiling FGF/FGFR TACC2 report-in-hand CCDC6 (N = 1,206) † AHCYL1 (N = 85) SHROOM3 TRIM8 TACC1 SLMAP PAWR FGFR2+ FGFR2+ NRAP (n = 74) (n = 64) NOL4 MACF1 FILIP1 CCDC170 Excluded ARHGAP24 (n = 31)* Enrolled in AFF4 N-of-One‡ cohort A (n = 107) 0% 10% 20% 30% Frequency of FGFR2 partner

C 80 D 50% FGFR2 FGFR2-rearranged Non–FGFR2-rearranged 60 40%

30% 40 20% Frequency

Number of patients 20 10%

0 ¶ AT M IDH1 TP53

12345678910111213141516171819202122X MLL3 FRS2 BAP1 TERT PTEN BRAF KRAS MDM2 ARID 2 ERBB 2 CCND 1 PBRM 1 PIK3CA ARID1A FGFR2

Chromosome CDKN2A CDKN2B

Figure 2. Genomic analysis of FGFR2-rearranged cholangiocarcinoma. A, Prescreening and cohort diagram. B, Frequency of FGFR2 rearrangement partners. C, Distribution of FGFR2 rearrangement partners throughout the genome. D, Frequency of co-occurring alterations in FGFR2-rearranged (blue) versus non–FGFR2-rearranged (yellow) patient samples. The FGFR2 bars represent patients with FGFR2 alterations other than rearrangements. *, 31 FGFR2+ patients not meeting other eligibility criteria were excluded. †, Reflects patients with a signed informed-consent form (none were excluded); 21 patients with report-in-hand were enrolled in cohorts B/C or other. ‡, Indicates 1 patient (0.71%) each with ACLY, ARHGAP22, ATAD2, ATF2, BICD1, CCDC158, CDC42BPB, CEP128, COL16A1, CTNNA3, DBP, DNAJC12, EEA1, EIF4ENIF1, ERC1, GAB2, GOPC, INSC, KCTD1, KIAA1598, MATR3, MCU, NEDD4L, NRBF2, PAH, POC1B, PXN, RABGAP1L, RASSF4, ROBO2, RPAP3, SFI1, SOGA1, SPICE1, STRN4, TBC1D1, TCTN3, TFEC, TTC28, TXLNB, USH2A, VCL, WAC, WDHD1, ZMYM4, and ZNF521 rearrangement partners. ¶, Nonrearrangement alterations only.

Patients with FGFR2 rearrangements had fewer GAs (3.7 the prescreening, alterations in IDH1 were less frequent in alterations/patient) than patients without FGFR2 rearrange- FGFR2-rearranged patients (5.1% vs. 10.7%), but were not ments (4.7 alterations/patient). Among FGFR2-rearranged significantly mutually exclusive when corrected for multi- versus non–FGFR2-rearranged patients, the most frequent ple testing (Fisher exact test; q value = 0.98; Supplementary co-alterations were BAP1 (38.4% vs. 8.2%), CDKN2A (21.7% Table S6). vs. 30.0%), CDKN2B (15.2% vs. 20.2%), and PBRM1 (9.4% vs. 10.7%; Fig. 2D). Similar to what was observed in our pre- Genomic Correlates of Response to Pemigatinib screening analysis, TP53 and KRAS alterations were observed We sought to interrogate the relationship between clinical less frequently in FGFR2-rearranged patients compared with response to pemigatinib and underlying molecular profiles. patients without FGFR2 rearrangements (TP53, 8.0% vs. 41.9%; A total of 107 centrally confirmedFGFR2 -rearranged patients KRAS, 1.4% vs. 24.2). Notably, 63.0% of FGFR2-rearranged were enrolled into cohort A of FIGHT-202. We first asked patients had co-alterations in a well-known tumor-suppres- whether the biomarker classification ofFGFR2 as a rearrange- sor gene, including BAP1, CDKN2A/B, TP53, PBRM1, ARID1A, ment or fusion had any impact on response to pemigatinib or PTEN. This was significantly lower than in patients with- (Table 1). Of 107 patients with FGFR2 fusions or rearrange- out FGFR2 rearrangements, of which 74.7% had alterations in ments, 92 were predicted to be fusions and 15 were classified tumor-suppressor genes (Fisher exact test; P = 0.0043), reflect- as rearrangements only. No significant difference in ORR was ing the lower number of co-alterations observed in FGFR2- observed for patients with FGFR2 alterations classified as rear- rearranged patients. Consistent with what was observed in rangements versus for patients with predicted fusions (40.0% vs.

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Table 1. Relationship between GAs and clinical outcomes

Group (n) ORR (%) OR (95% CI), P value Median PFS (95% CI), months PFS P value FGFR2+ population (107) 35.5 — 6.9 (6.2–9.6) — Alteration classification Fusion (92) 34.8 1.25 (0.4–3.8), 0.70 7.0 (6.0–10.5) 0.79 Rearrangement (15) 40.0 6.9 (4.7–11.7) FGFR2 partner Non-BICC1 (76) 36.8 0.82 (0.3–2.0), 0.65 9.0 (6.2–11.1) 0.20 BICC1 (31) 32.3 6.8 (2.6–8.9) Tumor suppressor Unaltered (43) 37.2 0.88 (0.4–2.0), 0.76 11.7 (9.1–17.4) 0.0003 Altered (64) 34.4 6.8 (4.9–6.9) BAP1 Unaltered (68) 30.9 1.7 (0.8–3.9), 0.19 9.1 (6.2–11.7) 0.06 Altered (39) 43.6 6.9 (4.7–8.9) CDKN2A/B Unaltered (86) 38.4 0.50 (0.2–1.5), 0.22 9.0 (6.4–11.1) 0.03 Altered (21) 23.8 6.4 (1.7–6.9) PBRM1 Unaltered (97) 36.1 0.76 (0.2–3.1), 0.70 7.0 (6.8–10.5) 0.05 Altered (10) 30.0 4.7 (1.4–10.8) TP53 Unaltered (98) 38.8 —a 9.0 (6.8–11.1) 0.0003 Altered (9) 0 2.8 (1.4–6.8) PIK3CA Unaltered (98) 35.7 0.90 (0.2–3.8), 0.89 8.8 (6.4–10.5) 0.10 Altered (9) 33.3 5.2 (1.5–11.1) IDH1 Unaltered (102) 36.3 0.44 (0.05–4.1), 0.47 6.9 (6.1–9.6) 0.28 Altered (5) 20.0 NE (1.4–NE)

Abbreviation: NE, not estimated. a Model did not converge.

34.8%, P = 0.70). Furthermore, median PFS was similar between to the small numbers of patients with any specific rearrange- these two populations (6.9 months vs. 7.0 months, P = 0.79). ment partner other than BICC1. Due to the large number (n = 63) of unique rearrangement Co-occurring alterations may be a mechanism of primary partners, we chose to compare the response to pemigatinib resistance to pemigatinib. We interrogated whether co-occur- in patients with the most frequent rearrangement partner, ring GAs had any impact on response to pemigatinib (Fig. 4A FGFR2–BICC1 (n = 31), with all others (n = 76; Fig. 3A). There and Table 1). The gene with the most frequent co-occurring was no significant difference in ORR (32.3% vs. 36.8%,P = GAs (BAP1) did not have a consistent impact on response cri- 0.65) or median PFS (6.8 months vs. 9.0 months, P = 0.20) teria. ORR was higher in BAP1-altered versus BAP1-unaltered in BICC1 versus all other partners (Fig. 3B and Table 1). No patients but did not reach statistical significance (43.6% vs. other comparisons of rearrangement partners were made due 30.9%, P = 0.19). In contrast, BAP1-altered patients trended

AB 60% Product-limit estimates With number of patients at risk and 95% confidence limits 40% 1.0 + Censored Log-rank P = 0.2022 20% 0.8 0% 0.6 −20% 0.4 −40% 0.2

−60% Survival probability 0.0 −80% BICC1 No Yes baseline in target lesion siz e

Best percentage change from FGFR2 partner BICC1 Non-BICC1 −100% 0510 15 20 25 PFS, months N 76 45 16 8 2 0 Y 31 18 6 2 0

Figure 3. Clinicogenomic analysis of FGFR2 rearrangement partners. A, Waterfall plot showing best percentage change from baseline in target lesion size following pemigatinib treatment. Blue bars correspond to rearrangements involving BICC1, and green corresponds to non-BICC1 rearrangements. B, Kaplan–Meier plot showing PFS in patients with BICC1 rearrangements (blue line) and non-BICC1 rearrangements (green line).

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AB 100% Best response Product-limit estimates 50% CR With number of patients at risk and 95% confidence limits PR 1.0 + Censored 0% **** Log-rank P = 0.0003 SD 0.8 −50% PD 0.6 NE −100% 0.4 100% FGFR2 0.2

Alteration Survival probability 37% BAP1 0.0 TP53 No Yes Loss 20% CDKN2A 0510 15 20 25 13% CDKN2B Amplification PFS, months N 98 60 22 10 20 9% PBRM1 Fusion Y 930 8% TP53 Rearrangement C 8% MYC Product-limit estimates Truncation With number of patients at risk and 95% confidence limits 8% PIK3CA Missense 1.0 + Censored Log-rank P = 0.0003 7% MCL1 Splice-site 0.8 5% IDH1 Frameshift 0.6 Nonsense 0.4 5% PTEN In-frame indel 0.2 3% FANCG

Survival probability 0.0 3% FOXP1 Tumor suppressor alteration No Yes

3% LRP1B 0510 15 20 25 PFS, months 3% LYN N 43 27 13 7 2 0 Y 64 36 9 3 0

Figure 4. Clinicogenomic analysis of genomic co-alterations. A, Oncoprint analysis of cohort A, ordered by magnitude of tumor shrinkage in evaluable patients. One patient had co-occurring FGFR2 missense mutations (see acquired resistance) due to confirmatory sample being collected at progression. B, Kaplan–Meier plot showing PFS in patients with TP53 alterations (blue line) and those without TP53 alterations (green line). C, Kaplan–Meier plot showing PFS in patients with tumor-suppressor alterations (blue line) and those without tumor-suppressor alterations (green line). CR, complete response; NE, not estimated; PD, progressive disease; PR, partial response; SD, stable disease. *, Represents nonevaluable patients (patients with no post-baseline tumor assessment). numerically toward shorter median PFS (6.9 months vs. A small number of patients had co-occurring alterations in 9.1 months, P = 0.06). Patients with GAs in CDKN2A/B or other known oncogenic driver genes, including PIK3CA (n = 9) PBRM1 alterations trended toward a lower ORR (CDKN2A/B, and IDH1 (n = 5). PIK3CA, which is downstream of FGFR2, 23.8% vs. 38.4%, P = 0.22; PBRM1, 30.0% vs. 36.1%; P = has been hypothesized to participate in signaling networks 0.70), although not statistically significant, and significantly mediating primary resistance to FGFR inhibitors. However, shorter median PFS (CDKN2A/B, 6.4 months vs. 9.0 months, no significant difference betweenPIK3CA -altered and PIK3CA- P = 0.03; PBRM1, 4.7 months vs. 7.0 months, P = 0.05). Of unaltered patients was observed in ORR (33.3% vs. 35.7%, P = note, patients with TP53 alterations (n = 9) had no objective 0.89) or in median PFS (5.2 months vs. 8.8 months, P = 0.1; responses and significantly shorter median PFS (2.8 months Table 1). A partial response was seen in 1 patient harboring an vs. 9.0 months, P = 0.0003; Table 1 and Fig. 4B). However, IDH1 alteration; however, the limited number of patients with 6 patients had stable disease, 3 with PFS >6 months, 2 patients co-occurring IDH1 mutations in this study makes it impos- experienced tumor shrinkage of 50% and 33%, and only 1 patient sible to assess the impact of these mutations on median PFS. had tumor growth >20%. Loss of different tumor-suppressor genes may play over- Response to Pemigatinib in Patients without lapping roles in tumor development. Therefore, we asked FGFR2 Fusions/Rearrangements whether patients with alterations in at least one well-known In FIGHT-202, an additional 20 patients with other FGF tumor suppressor, including BAP1, CDKN2A/B, TP53, or FGFR alterations were enrolled to cohort B (13 identified PBRM1, ARID1A, or PTEN, had a different response from by prescreening and 7 with report-in-hand), and 18 patients patients without tumor-suppressor loss. We found no signifi- without FGF or FGFR alterations were enrolled to cohort C cant difference in the ORR between patients with alterations (5 identified by prescreening and 13 with report-in-hand; in tumor-suppressor genes versus those without alterations Supplementary Fig. S4A). One patient without FGF or FGFR (34.4% vs. 37.2%, P = 0.76); however, we observed significantly alterations was enrolled in cohort C based on a local report, shorter median PFS (6.8 months vs. 11.7 months, P = 0.0003) but did not have sufficient sample material for confirma- in patients with tumor-suppressor gene loss as compared tion by FoundationOne (data for 17 subjects are therefore with those with no tumor-suppressor gene loss (Fig. 4C). presented). No independent centrally confirmed objective

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Genomic Profiling in FGFR2-Rearranged Cholangiocarcinoma RESEARCH ARTICLE responses were observed outside of cohort A, and median leading to less favorable pemigatinib-binding conditions. PFS was shorter in cohort B [2.1 (95% CI, 1.2–4.9) months] Collectively, these data demonstrate that acquired resistance and cohort C [1.7 (95% CI, 1.3–1.8) months]; however, several to pemigatinib mirrors that of other FGFR inhibitors in patients achieved stable disease, and tumor shrinkage was FGFR2-rearranged cholangiocarcinoma. noted in a subset of patients (22). To identify additional genomic subsets of patients for whom pemigatinib may elicit antitumor activity, we exam- DISCUSSION ined the GAs in these patients without FGFR2 fusions or By using genomic profiling to prescreen patients for rearrangements (Supplementary Fig. S4B and S4C). Most enrollment in FIGHT-202, we compiled a large database of notably, 3 of the 4 patients treated with pemigatinib who had mutational profiles for patients with cholangiocarcinoma. FGFR2 p.C382R mutations achieved a best overall response Given the rarity of cholangiocarcinoma, this information of stable disease, with a PFS of 6.9, 4.0, and 9.0 months. Two provides valuable insights into the broad mutational spec- of these patients had tumor shrinkage (−26.0% and −30.6%, trum of patients with this disease. Mutations in known unconfirmed) prior to disease progression. All 4 patients with tumor suppressors were widespread and most prevalent the FGFR2 p.C382R mutation had co-occurring BAP1 altera- in TP53, CDKN2A/B, ARID1A, SMAD4, and PBRM1. KRAS tions. We also observed stable disease in 3 of 6 patients with was the most frequently altered oncogene; however, KRAS FGF3/4/19 and CCND1 co-amplifications, with 2 patients p.G12C accounted for <5% of KRAS alterations. With the achieving tumor shrinkage (−32.5% and −41.4%, uncon- recent development of KRAS p.G12C inhibitors, this small firmed). One of 8 patients withFRS2 amplifications achieved population may benefit from targeted therapies. We esti- stable disease. In cohort C, tumor shrinkage was observed in mated the frequency of actionable alterations in patients 3 patients, but no unifying genomic features could be identi- with cholangiocarcinoma to be approximately 45%, pre- fied. Although only a small number of patients with each dominantly driven by alterations in IDH1/2, ERBB2, FGFR2, specific alteration type were treated in these cohorts, these PIK3CA, and BRAF. However, other potentially actionable data offer the potential for activity of pemigatinib in patients alterations were detected in <3% of patients each, including with other FGF/FGFR pathway alterations. BRCA1/2, FGFR3, FGFR1, KRAS, MET, ALK, RET, and ROS1. Notably, no NTRK1–3 rearrangements were identified in this Acquired Resistance to Pemigatinib cohort, despite previous reports of identification and clini- We performed genomic profiling on 8 patients with ini- cal benefit for NTRK-targeted therapeutics in patients with tial tumor shrinkage followed by progressive disease using NTRK+ cholangiocarcinoma (33, 34). We also found a small available tumor tissue (n = 6) or plasma (n = 2; Table 2); number of patients with MSI-H tumors who may benefit scans are provided for 5 patients (Supplementary Fig. S5). from inhibition of the PD-1/PD-L1 axis. Collectively, these Strikingly, every patient analyzed had at least one acquired data suggest that a substantial proportion of patients with mutation in FGFR2. All mutations were located in the FGFR2 cholangiocarcinoma may benefit from molecularly targeted kinase domain and were previously identified as resistance therapy in a disease with no currently approved second-line mutations to pemigatinib or other FGFR inhibitors (28–30). therapies. In total, we observed six unique mutations spanning five In addition to FGFR2, clinical trials for molecular tar- amino acid residues. FGFR2 p.N549K/H was observed in geted agents are ongoing for other alterations in cholan- 4 patients, whereas FGFR2 p.E565A, p.K659M, p.L617V, and giocarcinoma, including agents targeting IDH1. Patients p.K641R were each observed in 2 patients. Polyclonal resist- with cholangiocarcinoma have been enrolled in basket trials ance, defined by the presence of multiple acquired alterations for NTRK1–3 inhibitors and anti–PD-1 antibodies, which in the same patient, was identified in 3 patients, whereas only formed the basis for tumor-agnostic approvals for larotrec- a single mutation was observed in 5 other patients. tinib, entrectinib, and pembrolizumab. Furthermore, patients We performed in silico structural modeling to further under- with cholangiocarcinoma were included in other basket trials, stand the relationship between identified acquired resistance including a phase II trial of vemurafenib in patients with mutations in the kinase domain and pemigatinib binding BRAF p.V600E mutations (35). (Fig. 5A and B). Similar to previous reports with other FGFR Analysis of co-alteration patterns revealed that the strong- inhibitors, pemigatinib is in close contact with the FGFR2 est association between gene pairs was between FGFR2 and p.V564 gatekeeper residue. FGFR2 p.N549, p.K641, and BAP1. BAP1 is a deubiquitinase that forms the catalytic p.E565 contribute to a molecular brake that keeps the kinase subunit of the polycomb-repressive deubiquitinase complex in an inactive conformation (31). Mutations that disrupt involved in chromatin remodeling and is a bona fide tumor hydrogen bonding between any of these amino acids lead to a suppressor frequently mutated in mesothelioma, renal cell conformational shift and constitutive kinase activation. The carcinoma, and uveal melanoma (36–38). Jain and colleagues FGFR2 p.K659 residue is important for stabilizing the inac- previously reported BAP1 as the most frequently co-altered tive conformation of the activation loop (32). Thus, muta- gene with FGFR in cholangiocarcinoma and observed no tion of this residue also leads to kinase activation. FGFR2 association with overall survival (39). Consistent with their p.L617 resides in the hydrophobic spine and interacts with data, we did not observe a statistically significant differ- the phenylalanine residue of the “DFG” motif, which stabi- ence in ORR or median PFS for BAP1-altered versus BAP1- lizes a motif conformation conducive to binding of FGFR unaltered patients treated with pemigatinib. Furthermore, inhibitors (28). For example, mutation of FGFR2 p.L617 to Jain and colleagues observed an association of patients with a valine strengthens the hydrophobic spine of the kinase, co-occurring TP53 or CDKN2A/B alterations with shorter

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splice c site, ZWINT site, STAG2 p.M148fs*3 p.F135Lfs*40 BAP1 p.Q393 * BAP1 p.Q684 * TP53 p.R280G, splice FANCG site BIRC6 BAP1 splice PTEN p.T319fs*1 CDKN2A/B loss, None CTNNB1 p.S45C, KDR p.P1243L, p.K641R (23.7%) p.L617V (0.32%), p.L617V p.K659M (2.71%) p.N549K (4.03%) p.N549K (14.29%) p.E565A (6.1%), p.N549H (0.12%) p.K659M (6.07%) None p.N549K (23.19%) Acquired FGFR2 alterations (allele frequency) Co-alteration(s) p.E565A (0.19%), p.K641R (3.50%), p.L617V (33.9%) p.L617V Progression fusion fusion fusion rearrangement fusion fusion N/A N/A FGFR2–ATAD2 FGFR2–KIAA1217 FGFR2 rearrangement FGFR2–NRBF2 FGFR2–TRIM8 FGFR2–CCDC170 FGFR2-AHCYL1 b b Analysis platform WES F1L F1 F1 F1 F1 WES p.M148fs*3 loss, PTEN loss STAG2 Amp BAP1 p.Q684 *, AKT Amp, MDM4 PIK3C2B Amp p.R280G, FANCG splice site SH2B3 p.A227fs*51, FAS BAP1 splice site, MCL1 Amp, NTRK1 None BAP1 p.Q393 * EPHB6 p.R125Q F1L p.E545K, PIK3CA KDR p.P1243L, TP53 BRD4 p.Q256fs*46, Baseline fusion fusion fusion fusion rearrangement fusion fusion fusion FGFR2–PAWR FGFR2–ATAD2 FGFR2–KIAA1217 FGFR2 rearrangement Co-alteration(s) FGFR2–NRBF2 FGFR2–TRIM8 FGFR2–CCDC170 FGFR2-AHCYL1 FGFR2–WDHD1 Analysis platform MSKI F1 F1 F1 F1 ) F1 a ) F1 ) F1 a a −15.7 SD 13.1 −13 SD 8.8 (censored −39.8 PR 6.9 (censored −46 PR 9.1 −58.2 PR 6.9 −60.5 PR 15.9 (censored −33.3 SD 6.8 −63.6 PR 8.8 Tumor Tumor change from baseline, % BOR PFS, months rearrangements treated with pemigatinib Identification of acquired resistance mutations in patients with FGFR2 rearrangements able 2. fusions/rearrangements are not detectable in whole-exome sequencing data. are not detectable in whole-exome sequencing. FGFR2 fusions/rearrangements used from whole-exome Mutation data only were Censored at the time of the original data cutoff of March 22, 2019 (22), but progressed afterward. Censored at the time of original data cutoff March 22, 2019 (22), but progressed Not present in the FoundationOne panel. Not present in the FoundationOne Patient # 1 7 6 8 3 2 4 5 Abbreviations: BOR, best objective response; PR, partial response; SD, stable disease; MSKI, MSK Impact; F1, FoundationOne; F1L, FoundationOne Liquid; WES, whole exome sequencing. WES, whole exome Liquid; F1L, FoundationOne response; PR, partial SD, stable disease; MSKI, MSK Impact; F1, FoundationOne; BOR, best objective Abbreviations: a b c T

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V564 V564

E565 N549 E565 N549 K641 K659 L617 K641

L617

Figure 5. In silico analysis of acquired resistance mutations in FGFR2. Full (A) and zoomed-in (B) view of the FGFR2 ATP-binding pocket interac- tion with pemigatinib (orange). Important kinase domain regions are highlighted; gold, hinge region; red, catalytic loop; blue, activation domain; purple, c-alpha-helix; green, P-loop; cyan, DFG motif. Relevant amino acid residues are shown as colored ball and stick models. overall survival (39). Our results echo these results, finding to occur without an identified partner gene. The diversity in that patients with TP53, CDKN2A/B, and PBRM1 alterations FGFR2 rearrangement partners has been reported by previous had shorter median PFS on pemigatinib than those without studies (13, 15, 26, 40). For FGFR2, the mechanism of activa- alterations in these genes. However, the small number of tion is unique from most other kinase fusions in that FGFR2 patients, particularly with TP53 or PBRM1 alterations, must fusions are expressed by the endogenous FGFR2 promoter be acknowledged as a limitation. In this study, we advanced and include the majority of the protein. In general, the final these prior efforts by grouping alterations in known tumor- exon (exon 18), corresponding to the cytoplasmic post-kinase suppressor genes, including BAP1, CDKN2A/B, TP53, PBRM1, region, is replaced with sequences from the partner gene. ARID1A, or PTEN. This group of patients, accounting for Although the most prevalent FGFR2 partner (BICC1) has a 63% of all patients, had significantly shorter median PFS, dimerization domain, not all FGFR2 rearrangement partners although ORR was similar between the two groups. It should have known or predicted dimerization domains, suggest- be noted, however, that responses or prolonged stable disease ing that the pervasiveness of FGFR2 partners may be due to has been observed in individual patients within this group. lack of requirement for sequence or functional specificity of Collectively, these data suggest that patients with specific co- partner genes. Importantly, we saw no differences in clinical occurring alterations, especially in tumor-suppressor genes, response to pemigatinib in patients with BICC1 versus other may have worse outcomes with FGFR inhibitors. Additional partners. strategies, such as combination therapies, may be particularly DNA-based assays, such as FoundationOne, resolve the warranted in this patient population. genomic breakpoints of the rearrangement and do not inter- The co-occurrence and mutual exclusivity analysis also rogate the fusion at the level of RNA or protein. Therefore, revealed that although IDH1 alterations were depleted in rearrangements not classified as fusions, including those patients with FGFR2 rearrangements, they were not statisti- with no identified partner gene, may express fusion proteins cally mutually exclusive. This was surprising given the known by utilizing posttranscriptional controls, such as alternative role of both FGFR2 and IDH1 as oncogenic drivers in intra- splicing, or be generated through more complex genomic hepatic cholangiocarcinoma and in contrast to previous rearrangements involving multiple breakpoints, termed reports of mutual exclusivity between FGFR2 and IDH1 (13). “bridged fusions” (41). Alternatively, these events may repre- Of 5 patients with both FGFR2 rearrangements and IDH1 sent truncation of FGFR2 in the post-kinase region. FGFR2 alterations enrolled in FIGHT-202, 1 had a partial response, truncation has been shown to be sufficient to drive ligand- suggesting that FGFR inhibitors can have activity in the pres- independent growth and may represent an alternative mecha- ence of both alterations; however, more data are required to nism of FGFR2 activation, distinct from fusions (42–44). determine if the IDH1 alterations affect median PFS. As both FGFR2 truncations may be missed by RNA-based approaches FGFR and IDH1 inhibitors are under clinical evaluation that are not designed to identify truncating rearrangements. in cholangiocarcinoma, it will be important to study how Detailed analysis of these FGFR2 rearrangements, on both this subset of patients responds to single or combination DNA- and RNA-based platforms, will be necessary to resolve therapies. these questions. We saw no differences in clinical outcomes In this study, we identified a large number of partner for patients with FGFR2 rearrangements versus fusions and genes for FGFR2; in 138 patients with FGFR2 rearrangements did see responses in patients with FGFR2 rearrangement with profiled, we identified 63 uniqueFGFR2 partner genes. Fur- no identified partner gene. Due to the small sample size, it is thermore, 27 patients were classified as having only anFGFR2 not possible to suggest the functional equivalency of fusions rearrangement, and 13 of these rearrangements were reported and rearrangements.

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Although no objective responses were observed in patients oped both the FGFR2 p.E565A and p.L617M mutations (49). treated with pemigatinib without FGFR2 rearrangements or These investigators performed in vitro analysis of these muta- fusions, and median PFS was shorter in these patients, post hoc tions in three different cell lines and observed potency shifts genomic subset analysis identified interesting patterns that to several selective FGFR inhibitors including infigratinib, may warrant further investigation. We noted stable disease in AZD4547, and erdafitinib. The only mutation identified in 3 of 4 patients with the FGFR2 p.C382R mutation (2 of whom the current study that has not been profiledin vitro is FGFR2 had tumor shrinkage prior to disease progression), which is p.K641R. Given the role of FGFR2 p.K641 in contributing to a known oncogenic mutation in the extracellular domain the molecular brake (along with p.N549 and p.E565), this of FGFR2 that activates FGFR signaling (45). Interestingly, mutation is expected to similarly affect pemigatinib function. all 4 patients with FGFR2 p.C382R mutations had co-occur- Although pemigatinib was not specifically evaluated in these ring BAP1 alterations. Given the strong enrichment of BAP1 later in vitro studies, the shared structural features of FGFR alterations in FGFR2-rearranged cholangiocarcinoma, this inhibitors combined with shared clinical resistance profiles may suggest FGFR2 p.C382R and FGFR2 rearrangements provide compelling evidence to support the identifiedFGFR2 have a similar molecular etiology. FGFR inhibitors have mutations as contributing to resistance. shown clinical efficacy in other cancer types withFGFR muta- Polyclonal resistance, involving multiple acquired FGFR2 tions, most notably in FGFR3-mutated urothelial carcinoma mutations, was observed in 3 patients. This finding is reminis- (46). Although activating FGFR2 mutations are rare in chol- cent of previous evidence from other groups demonstrating angiocarcinoma, future tumor-agnostic studies with FGFR polyclonal acquired resistance in patients with FGFR2- inhibitors, such as the FIGHT-207 study (NCT03822117) of rearranged cholangiocarcinoma treated with infigratinib or pemigatinib, will investigate the role of FGFR inhibition in Debio1347 (28, 29, 50). Collectively, these findings suggest a FGFR2-mutated cholangiocarcinoma. high level of tumor heterogeneity and a strong selective pres- We observed stable disease in 3 of 6 patients treated with sure by FGFR inhibitors. pemigatinib harboring the FGF3/4/19 and CCND1 amplifica- Interestingly, no gatekeeper (FGFR2 p.V564) mutations tions (2 patients with tumor shrinkage). FGF3/4/19 are signal- were identified in this study as has been reported with other ing ligands that bind with varying specificities to the different FGFR inhibitors (28, 29). Differences in mutational resist- FGF receptors to promote dimerization and transphosphoryl- ance profile are likely due to differences in drug-binding ation (47). FGF3/4/19 and CCND1 are co-located on the same specificities. Further study into the exact resistance profile chromosomal region (11q13) and are often co-amplified. for all FGFR inhibitors is needed to ensure appropriate treat- Of note, Jain and colleagues also described a patient with ment order for patients who acquire resistance. There is a cholangiocarcinoma harboring an FGF19 amplification who need for FGFR inhibitors whose potency is not affected by was treated with an FGFR inhibitor for 1 year and had a sta- these kinase domain mutations, including the gatekeeper ble response to treatment (39). Just 1 of 8 patients with FRS2 mutations. amplifications achieved stable disease. FRS2 is an adapter Although these data offer useful insights into the overall protein that links FGFR signaling to the MAP kinase pathway molecular landscape of patients with cholangiocarcinoma, (48). Stable disease was observed in 3 patients in cohort C, it is important to consider the limitations of our study. but no unifying genomic features were identified. Additional Patients prescreened for FIGHT-202 included those with investigation into the role of pemigatinib in patients without both intrahepatic and extrahepatic cholangiocarcinoma sub- FGFR fusions or rearrangements is warranted. However, these types, location not being captured in the clinical database. data suggest a requirement for strong activation of the FGFR Cholangiocarcinoma subtypes are known to harbor distinct pathway, which may be restricted to FGFR rearrangements, mutational profiles, and thus estimation of actionable altera- fusions, and possibly activating mutations. tion frequencies in specific cholangiocarcinoma subtypes is Consistent with previous reports of resistance to other not possible with these data (26). For example, the frequency FGFR inhibitors in FGFR2-rearranged cholangiocarcinoma, of FGFR2 rearrangements (6.1%) is lower than previously we found acquired FGFR2 mutations in all 8 patients ana- reported for pure intrahepatic cholangiocarcinoma popu- lyzed at progression (28–30). FGFR2 p.N549H was identified lations (10%–16%), likely due to the inclusion of patients as a resistance mechanism to pemigatinib in a patient with with extrahepatic cholangiocarcinoma (typically negative cholangiocarcinoma harboring an FGFR2–CLIP1 fusion, and for FGFR2 rearrangements) in the prescreening population. was shown to shift the potency of selective FGFR inhibitors, Patients may have been previously prescreened for other including pemigatinib, AZD4547, infigratinib, and erdafi- clinical trials, and thus patients positive for clinical trial bio- tinib, in NIH3T3 cells expressing the FGFR2–CLIP1 fusion markers, including FGFR2 and IDH1, may not be accurately (30). Furthermore, all 6 acquired FGFR2 mutations identified represented in our cohort. Finally, regional differences in in this study were previously reported by Goyal and col- the availability of genomic profiling may have significantly leagues in their studies of patients with FGFR2-rearranged skewed the purity of the cholangiocarcinoma population pre- cholangiocarcinoma who progressed on either infigratinib screened in different parts of the world. Genomic testing is or Debio1347 (29). These investigators demonstrated that regularly performed on patients with cholangiocarcinoma in CCLP-1 cells engineered to express the FGFR2–PHGDH fusion the United States, but less frequently in Europe and the rest with a series of these resistance mutations shifted the potency of the world. Thus, we saw large differences in the frequency of infigratinib and Debio1347 to varying levels. In a second of FGFR2 alterations by region (15.2% in United States, 7.4% report, Krook and colleagues identified anFGFR2–KIAA1598 in Europe, and 2.2% in the rest of the world), which may be fusion–positive patient treated with infigratinib who devel- due to inclusion of extrahepatic and noncholangiocarcinoma

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Genomic Profiling in FGFR2-Rearranged Cholangiocarcinoma RESEARCH ARTICLE cancer subtypes. Consistent with the potential higher frac- ease progression. The present analysis investigated the relationship tion of nonintrahepatic cholangiocarcinoma in Asia versus between genomic features and clinical outcomes in patients with Europe and the United States, higher rates of TP53, CDKN2A, cholangiocarcinoma harboring FGFR2 alterations receiving pemi- and KRAS were observed in patients from the rest of the world gatinib in FIGHT-202. versus Europe and the United States and possibly reflect a Genomic Analysis difference in prevalence of extrahepatic versus intrahepatic cholangiocarcinoma rather than regional differences. Future Archival, formalin-fixed, paraffin-embedded tumor samples from studies should collect detailed clinical data on patients with all prescreened or enrolled patients were analyzed for GAs using the FoundationOne-targeted next-generation DNA-sequencing assay cholangiocarcinoma around the world and analyze patients (Foundation Medicine Inc.), which uses hybrid capture–based DNA using the same genomic analysis platform. target enrichment to identify somatic GAs in the coding regions of In conclusion, our study highlights the utility of genomic 315 cancer-related genes and introns from 28 genes often rearranged profiling in the context of molecularly guided clinical trials. in cancer (12). The assay detects multiple classes of GAs including We utilized prescreening data from the FIGHT-202 clinical base substitutions, insertions and deletions, copy-number altera- trial to gain additional insights into the broad molecular tions, and rearrangements. Patients entering the study with an exist- profile of patients with cholangiocarcinoma. We further inves- ing FoundationOne report may have used a different version of the tigated the unique molecular features of patients with FGFR2- panel containing up to 404 genes. The FGFR2 content is the same, rearranged cholangiocarcinoma and interrogated the relation- independent of which assay version was used. Amino acid numbering ship between genomic profile and response to pemigatinib. for the FGFR2 gene was based on the RefSeq transcript NM_000141. Finally, utilizing additional NGS approaches, we identified Molecular Modeling mechanisms of acquired resistance to pemigatinib. Collec- tively, these data advance our understanding of patients with In silico modeling was performed using pemigatinib docked to cholangiocarcinoma, FGFR2 rearrangements, and mechanisms FGFR2 (PDB structure 1EOC.pdb) Molecular operating environ- ment (Chemical Computing Group Inc.). of primary and acquired resistance to pemigatinib. These data should serve as a foundation for the advancement of treatment Statistical Methods for patients with FGFR2-rearranged cholangiocarcinoma. Statistical analyses of genomic correlates of response, including FGFR2 rearrangement partner and co-occurring GAs, were performed METHODS using a log-likelihood ratio test. PFS distributions were estimated using the Kaplan–Meier method; statistical differences in PFS distri- Study Design butions in the presence or absence of FGFR2 alterations were calcu- Details on the study design, eligibility criteria, and efficacy and lated using the nonparametric log-rank test. Logistic regressions were safety findings of FIGHT-202 have been published previously (22). employed to compare ORR between groups. Statistical analyses were FIGHT-202 (NCT02924376), a phase II, open-label, multicenter, performed using SAS (Enterprise Guide 7.1) and R version 3.5.2 [R-3.5.2 global study of pemigatinib in patients with previously treated for Windows (32/64 bit); https://cran.r-project.org/bin/windows/ advanced or metastatic cholangiocarcinoma, with or without FGF/ base/old/3.5.2/]. FGFR alterations, was conducted at 146 sites in the United States, Europe, the Middle East, and Asia. Key eligibility criteria for enroll- Data Reporting ment in FIGHT-202 were as follows: age ≥18 years; confirmed diag- Researchers may request anonymized datasets from any interven- nosis of locally advanced or metastatic cholangiocarcinoma based on tional study (except phase I studies) for which the product and indica- histology/cytology; disease progression following ≥1 prior systemic tion have been approved on or after January 1, 2020, in at least one therapy (prior receipt of selective FGFR inhibitors was not allowed); major market (e.g., United States, Europe, and Japan). Information on Eastern Cooperative Oncology Group performance status ≤2; radio- Incyte’s clinical trial data sharing policy and instructions for submit- logically measurable disease per RECIST v1.1. ting clinical trial data requests are available at: https://www.incyte.com/ Before eligibility screening, patients were initially prescreened cen- Portals/0/Assets/Compliance%20and%20Transparency/clinical- trally for FGF/FGFR status using NGS (FoundationOne). Patients trial-data-sharing.pdf?ver=2020–05–21–132838–960 were also permitted to proceed to eligibility screening if they pos- sessed an existing FoundationOne report or an FGF/FGFR status report derived from a local assessment. Local documentation of Authors’ Disclosures FGF/FGFR status required retrospective central confirmation using I.M. Silverman reports other from Incyte Corporation outside the FoundationOne. Based on central results, patients were assigned to submitted work. A. Hollebecque reports grants from Incyte during cohorts A (FGFR2 fusions or rearrangements), B (other FGF/FGFR the conduct of the study, personal fees and other from Amgen, per- GAs), or C (no FGF/FGFR GAs). sonal fees from Eisai, personal fees and other from Servier, personal Regardless of cohort assignment, patients orally self-administered fees from QED Therapeutics, grants and other from AstraZeneca, pemigatinib at a starting dose of 13.5 mg once daily on a 21-day grants, personal fees, and other from Incyte, other from Lilly, per- (2-weeks-on, 1-week-off) cycle, until radiologic disease progression, sonal fees from Spectrum Pharmaceuticals, and other from Roche unacceptable toxicity, withdrawal of consent, or decision to discon- outside the submitted work. L. Friboulet reports grants from Incyte tinue treatment by the patient or physician. during the conduct of the study and grants from Debiopharm The primary endpoint of FIGHT-202 was ORR in cohort A per outside the submitted work. S. Owens reports other from Incyte independent central review; secondary endpoints included were ORR Corporation outside the submitted work. R.C. Newton reports being in cohorts B, A + B (all patients with FGF/FGFR alterations), and C; an employee of the Incyte Research Institute. L. Féliz reports other duration of response, disease control rate, PFS, overall survival, and from Incyte during the conduct of the study; other from Incyte out- safety in all cohorts. Tumor response was assessed by independent side the submitted work; and is an employee from Incyte and owns review according to RECIST v1.1; disease status was assessed until stocks. T.C. Burn reports other from Incyte Corporation outside the disease progression or discontinuation due to any reason except dis- submitted work. No disclosures were reported by the other authors.

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Authors’ Contributions 11. Ying J, Chen J. Combination versus mono-therapy as salvage treatment for advanced biliary tract cancer: a comprehensive meta-analy- I.M. Silverman: Conceptualization, data curation, formal analysis of published data. Crit Rev Oncol Hematol 2019;139:134–42. sis, visualization, writing-original draft, writing-review and editing. 12. Frampton GM, Fichtenholtz A, Otto GA, Wang K, Downing SR, He J, A. Hollebecque: Resources, investigation, writing-review and editing. et al. Development and validation of a clinical cancer genomic profil- L. Friboulet: Resources, investigation, writing-review and editing. ing test based on massively parallel DNA sequencing. Nat Biotechnol S. Owens: Data curation, formal analysis, visualization, writing- 2013;31:1023–31. review and editing. R.C. Newton: Supervision, writing-review and 13. Lowery MA, Ptashkin R, Jordan E, Berger MF, Zehir A, Capanu M, editing. H. Zhen: Data curation, supervision, methodology, writing- et al. Comprehensive molecular profiling of intrahepatic and extra- review and editing. L. Féliz: Supervision, methodology, writing-review hepatic cholangiocarcinomas: potential targets for intervention. Clin and editing. C. Zecchetto: Resources, investigation, writing-review Cancer Res 2018;24:4154–61. and editing. D. Melisi: Resources, investigation, writing-review and 14. Churi CR, Shroff R, Wang Y, Rashid A, Kang HC, Weatherly J, et al. editing. T.C. Burn: Conceptualization, supervision, methodology, Mutation profiling in cholangiocarcinoma: prognostic and therapeu- writing-review and editing. tic implications. PLoS One 2014;9:e115383. 15. Jain A, Javle M. Molecular profiling of biliary tract cancer: a target Acknowledgments rich disease. J Gastrointest Oncol 2016;7:797–803. 16. Graham RP, Barr Fritcher EG, Pestova E, Schulz J, Sitailo LA, Vas- This study was sponsored by Incyte Corporation. The authors matzis G, et al. Fibroblast growth factor receptor 2 translocations in wish to thank the patients and their families, the investigators, intrahepatic cholangiocarcinoma. Hum Pathol 2014;45:1630–8. and the site personnel who participated in this study. The authors 17. Ross JS, Wang K, Gay L, Al-Rohil R, Rand JV, Jones DM, et al. also thank Ravi Jalluri, PhD, for assistance with molecular mod- New routes to targeted therapy of intrahepatic cholangiocarcinomas eling. Editorial assistance was provided by Abigail Marmont, PhD, revealed by next-generation sequencing. Oncologist 2014;19:235–42. CMPP, of Envision Pharma Group, Inc., and funded by Incyte 18. Farshidfar F, Zheng S, Gingras MC, Newton Y, Shih J, Robertson AG, Corporation. Work in the Digestive Molecular Clinical Oncology et al. Integrative genomic analysis of cholangiocarcinoma identifies Research Unit, Università degli Studi di Verona, Italy, was partially distinct IDH-mutant molecular profiles. 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FEBRUARY 2021 CANCER DISCOVERY | OF14

Clinicogenomic Analysis of FGFR2-Rearranged Cholangiocarcinoma Identifies Correlates of Response and Mechanisms of Resistance to Pemigatinib

Ian M. Silverman, Antoine Hollebecque, Luc Friboulet, et al.

Cancer Discov Published OnlineFirst November 20, 2020.

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